Anti-TAT226 antibodies and immunoconjugates

ABSTRACT

Anti-TAT226 antibodies and immunoconjugates thereof are provided. Methods of using anti-TAT226 antibodies and immunoconjugates thereof are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/687,309, filed on Mar. 16, 2007 (now U.S. Pat. No. 8,183,347), whichclaims the benefit of U.S. Provisional Application No. 60/783,746, filedon Mar. 17, 2006, the disclosures of which are incorporated herein byreference in their entirety.

This application claims the benefit of U.S. Provisional Application No.60/783,746, filed Mar. 17, 2006, the disclosure of which is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to anti-TAT226 antibodies andimmunconjugates thereof. The invention further relates to methods ofusing anti-TAT226 antibodies and immunconjugates thereof.

BACKGROUND

Antibodies that bind to polypeptides expressed on the surface of cancercells have proven to be effective anti-cancer therapies. Such antibodiesact through various mechanisms including, for example, activation ofantibody-dependent cell-mediated cytotoxicity (ADCC); induction by theantibody of complement-dependent cytotoxicity (CDC); enhancement ofcytokine release; and induction of apoptosis. See, e.g, Cardarelli etal. (2002) Cancer Immunol. Immunother. 51:15-24. For example, HERCEPTIN®and RITUXAN® (both from Genentech Inc., South San Francisco, Calif.) areantibodies that have been used successfully to treat breast cancer andnon-Hodgkin's lymphoma, respectively. HERCEPTIN® is a recombinantDNA-derived humanized monoclonal antibody that selectively binds to theextracellular domain of the human epidermal growth factor receptor 2(HER2) proto-oncogene. HER2 protein overexpression is observed in 25-30%of primary breast cancers. RITUXAN® is a genetically engineered chimericmurine/human monoclonal antibody directed against the CD20 antigen foundon the surface of normal and malignant B lymphocytes. Both theseantibodies are recombinantly produced in CHO cells. HERCEPTIN® appearsto act, at least in part, by inhibiting angiogenesis (Izumi et al.(2002) Nature 416:279-280), and RITUXAN® appears to act, at least inpart, by inducing apoptosis (Cardarelli et al. (2002) Cancer Immunol.Immunother. 51:15-24).

Immunoconjugates, or “antibody-drug conjugates,” are useful for thelocal delivery of cytotoxic agents in the treatment of cancer. See,e.g., Syrigos et al. (1999) Anticancer Research 19:605-614;Niculescu-Duvaz et al. (1997) Adv. Drug Deliv. Rev. 26:151-172; U.S.Pat. No. 4,975,278. Immunoconjugates allow for the targeted delivery ofa drug moiety to a tumor, whereas systemic administration ofunconjugated cytotoxic agents may result in unacceptable levels oftoxicity to normal cells as well as the tumor cells sought to beeliminated. See Baldwin et al. (Mar. 15, 1986) Lancet pp. 603-05; Thorpe(1985) “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: AReview,” in Monoclonal Antibodies '84: Biological and ClinicalApplications (A. Pinchera et al., eds.) pp. 475-506. Immunoconjugatesthat target cell surface polypeptides have been and continue to bedeveloped for the treatment of cancer. For review, see, e.g., Hamann etal. (2005) Expert Opin. Ther. Patents (2005) 15:1087-1103.

It is clear that there is a continuing need for agents that target cellsurface polypeptides for diagnostic and/or therapeutic purposes. Theinvention described herein meets this need and provides other benefits.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

SUMMARY

The invention provides anti-TAT226 antibodies and methods of using thesame.

In one aspect, an antibody that binds to TAT226 is provided, wherein theantibody comprises at least one, two, three, four, five, or six HVRsselected from:

-   -   (1) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:4;    -   (2) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:5;    -   (3) an HVR-H3 comprising an amino acid sequence that conforms to        the consensus sequence of SEQ ID NO:11;    -   (4) an HVR-L1 comprising the amino acid sequence of SEQ ID        NO:12;    -   (5) an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:13; and    -   (6) an HVR-L3 comprising an amino acid sequence that conforms to        the consensus sequence of SEQ ID NO:19.

In another aspect, an antibody that binds to TAT226 comprises (a) anHVR-H3 comprising an amino acid sequence that conforms to the consensussequence of SEQ ID NO:11, and (b) at least one, two, three, four or fiveHVRs selected from:

-   -   (1) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:4;    -   (2) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:5;    -   (3) an HVR-L1 comprising the amino acid sequence of SEQ ID        NO:12;    -   (5) an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:13; and    -   (6) an HVR-L3 comprising an amino acid sequence that conforms to        the consensus sequence of SEQ ID NO:19.        In one embodiment, the antibody comprises an HVR-L3 comprising        an amino acid sequence that conforms to the consensus sequence        of SEQ ID NO:19. In one embodiment, the antibody further        comprises an HVR-H1 comprising the amino acid sequence of SEQ ID        NO:4 and an HVR-H2 comprising the amino acid sequence of SEQ ID        NO:5. In one embodiment, the antibody further comprises an        HVR-L1 comprising the amino acid sequence of SEQ NO:12 and an        HVR-L2 comprising the amino acid sequence of SEQ ID NO:13.

In one embodiment, the antibody comprises an HVR-H3 comprising an aminoacid sequence selected from SEQ ID NO:6-10. In one embodiment, theantibody further comprises an HVR-L3 comprising an amino acid sequenceselected from SEQ ID NO:14-18. In one embodiment, the HVR-H3 comprisesthe amino acid sequence of SEQ ID NO:9, and the HVR-L3 comprises theamino acid sequence of SEQ ID NO:17. In one embodiment, the HVR-H3comprises the amino acid sequence of SEQ ID NO:10, and the HVR-L3comprises the amino acid sequence of SEQ ID NO:18. In one embodiment,the antibody further comprises an HVR-H1 comprising the amino acidsequence of SEQ ID NO:4 and an HVR-H2 comprising the amino acid sequenceof SEQ ID NO:5. In one embodiment, the antibody further comprises anHVR-L1 comprising the amino acid sequence of SEQ NO:12 and an HVR-L2comprising the amino acid sequence of SEQ ID NO:13.

In one aspect, an antibody that binds to TAT226 is provided, wherein theantibody comprises at least one, two, three, four, five, or six HVRsselected from:

-   -   (1) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:1;    -   (2) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:2;    -   (3) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:3;    -   (4) an HVR-L1 comprising the amino acid sequence of SEQ ID        NO:12;    -   (5) an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:13; and    -   (6) an HVR-L3 comprising the amino acid sequence of SEQ ID        NO:14.

In another aspect, an antibody that binds to TAT226 comprises (a) anHVR-H3 comprising the amino acid sequence of SEQ ID NO:3, and (b) atleast one, two, three, four, or five HVRs selected from:

-   -   (1) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:1;    -   (2) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:2;    -   (3) an HVR-L1 comprising the amino acid sequence of SEQ ID        NO:12;    -   (4) an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:13; and    -   (5) an HVR-L3 comprising the amino acid sequence of SEQ ID        NO:14.        In one embodiment, the antibody comprises an HVR-L3 comprising        the amino acid sequence of SEQ ID NO:14. In one embodiment, the        antibody further comprises an HVR-H1 comprising the amino acid        sequence of SEQ ID NO:1 and an HVR-H2 comprising the amino acid        sequence of SEQ ID NO:2. In one embodiment, the antibody further        comprises an HVR-L1 comprising the amino acid sequence of SEQ        NO:12 and an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:13.

In certain embodiments, any of the above antibodies further comprises atleast one framework selected from a VH subgroup III consensus frameworkand a VL subgroup I consensus framework.

In one aspect, an antibody that binds to TAT226 is provided, wherein theantibody comprises a heavy chain variable domain having at least 90%sequence identity to an amino acid sequence selected from SEQ IDNO:21-25. In one embodiment, the antibody further comprises a lightchain variable domain having at least 90% sequence identity to an aminoacid sequence selected from SEQ ID NO:26-31. In one embodiment, theantibody comprises a heavy chain variable domain having at least 90%sequence identity to the amino acid sequence of SEQ ID NO:24. In oneembodiment, the antibody further comprises a light chain variable domainhaving at least 90% sequence identity to the amino acid sequence of SEQID NO:29. In one embodiment, the heavy chain variable domain comprisesthe amino acid sequence of SEQ ID NO:24, and the light chain variabledomain comprises the amino acid sequence of SEQ ID NO:29. In oneembodiment, the antibody comprises a heavy chain variable domain havingat least 90% sequence identity to the amino acid sequence of SEQ IDNO:25. In one embodiment, the antibody further comprises a light chainvariable domain having at least 90% sequence identity to the amino acidsequence of SEQ ID NO:30. In one embodiment, the heavy chain variabledomain comprises the amino acid sequence of SEQ ID NO:25, and the lightchain variable domain comprises the amino acid sequence of SEQ ID NO:30.

In one aspect, an antibody that binds to TAT226 is provided, wherein theantibody comprises a heavy chain variable domain having at least 90%sequence identity to the amino acid sequence of SEQ ID NO:20. In oneembodiment, the antibody further comprises a light chain variable domainhaving at least 90% sequence identity to the amino acid sequence of SEQID NO:26. In one embodiment, the heavy chain variable domain comprisesthe amino acid sequence of SEQ ID NO:20, and the light chain variabledomain comprises the amino acid sequence of SEQ ID NO:26.

In certain embodiments, a polynucleotide encoding any of the aboveantibodies is provided. In one embodiment, a vector comprising thepolynucleotide is provided. In one embodiment, a host cell comprisingthe vector is provided. In one embodiment, the host cell is eukaryotic.In one embodiment, the host cell is a CHO cell. In one embodiment, amethod of making an anti-TAT226 antibody is provided, wherein the methodcomprises culturing the host cell under conditions suitable forexpression of the polynucleotide encoding the antibody, and isolatingthe antibody.

In one aspect, an antibody that binds to TAT226 expressed on the surfaceof a cell is provided. In one embodiment, the antibody binds to anepitope within a region of TAT226 from amino acid 21-115 of SEQ IDNO:75. In one embodiment, the cell is a cancer cell. In one embodiment,the cancer cell is an ovarian cancer cell, a brain tumor cell, or aWilm's tumor cell.

In certain embodiments, any of the above antibodies is a monoclonalantibody. In one embodiment, the antibody is an antibody fragmentselected from a Fab, Fab′-SH, Fv, scFv, or (Fab′)₂ fragment. In oneembodiment, the antibody is humanized. In one embodiment, the antibodyis human. In one embodiment, the antibody binds to the same epitope asan antibody selected from YWO.32, YWO.49, YWO.49.B7, YWO.49.C9,YWO.49.H2, and YWO.49.H6.

In one aspect, a method of detecting the presence of TAT226 in abiological sample is provided, the method comprising contacting thebiological sample with any of the above antibodies under conditionspermissive for binding of the antibody to TAT226, and detecting whethera complex is formed between the antibody and TAT226. In one embodiment,the biological sample comprises ovarian tumor cells, brain tumor cells,or Wilms' tumor cells.

In one aspect, a method of diagnosing a cell proliferative disorderassociated with increased expression of TAT226 is provided, the methodcomprising contacting a test cell with any of the above antibodies;determining the level of expression of TAT226 by detecting binding ofthe antibody to TAT226; and comparing the level of expression of TAT226by the test cell with the level of expression of TAT226 by a controlcell, wherein a higher level of expression of TAT226 by the test cell ascompared to the control cell indicates the presence of a cellproliferative disorder associated with increased expression of TAT226.In one embodiment, the test cell is a cell from a patient suspected ofhaving a cell proliferative disorder. In one embodiment, the cellproliferative disorder is selected from ovarian cancer and Wilms' tumor.In one embodiment, the method comprises determining the level ofexpression of TAT226 on the surface of the test cell and comparing thelevel of expression of TAT226 on the surface of the test cell with thelevel of expression of TAT226 on the surface of the control cell.

The invention further provides immunoconjugates and methods of using thesame.

In one aspect, an immunoconjugate comprises any of the above anti-TAT226antibodies covalently attached to a cytotoxic agent. In one embodiment,the cytotoxic agent is selected from a toxin, a chemotherapeutic agent,an antibiotic, a radioactive isotope, and a nucleolytic enzyme.

In one aspect, an immunoconjugate having the formula Ab-(L-D)p isprovided, wherein:

-   -   (a) Ab is any of the above anti-TAT226 antibodies,    -   (b) L is a linker;    -   (c) D is a drug of formula D_(E) or D_(F)

-   -   -   and wherein R² and R⁶ are each methyl, R³ and R⁴ are each            isopropyl, R⁷ is sec-butyl, each R⁸ is independently            selected from CH₃, O—CH₃, OH, and H; R⁹ is H; R¹⁰ is aryl; Z            is —O— or —NH—; R¹¹ is H, C₁-C₈ alkyl, or            —(CH₂)₂—O—(CH₂)₂—O—(CH₂)₂—O—CH₃; and R¹⁸ is            —C(R⁸)₂—C(R⁸)₂-aryl; and

    -   (d) p ranges from about 1 to 8.        In one embodiment, the antibody (Ab) comprises 1) an HVR-H3        comprising an amino acid sequence that conforms to the consensus        sequence of SEQ ID NO:11 and 2) at least one, two, three, four,        or five HVRs selected from:

    -   (i) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:4;

    -   (ii) an HVR-H2 comprising the amino acid sequence of SEQ ID        NO:5;

    -   (iii) an HVR-L1 comprising the amino acid sequence of SEQ ID        NO:12;

    -   (iv) an HVR-L2 comprising the amino acid sequence of SEQ ID        NO:13; and

    -   (v) an HVR-L3 comprising an amino acid sequence that conforms to        the consensus sequence of SEQ ID NO:19.        In one embodiment, the antibody comprises an HVR-L3 comprising        an amino acid sequence that conforms to the consensus sequence        of SEQ ID NO:19. In one embodiment, the antibody comprises an        HVR-H3 comprising the amino acid sequence of SEQ ID NO:9 and an        HVR-L3 comprising the amino acid sequence of SEQ ID NO:17. In        one embodiment, the antibody comprises an HVR-H3 comprising the        amino acid sequence of SEQ ID NO:10 and an HVR-L3 comprising the        amino acid sequence of SEQ ID NO:18. In one embodiment, the        antibody further comprises an HVR-H1 comprising the amino acid        sequence of SEQ ID NO:4, an HVR-H2 comprising the amino acid        sequence of SEQ ID NO:5, an HVR-L1 comprising the amino acid        sequence of SEQ ID NO:12, and an HVR-L2 comprising the amino        acid sequence of SEQ ID NO:13. In one embodiment, the antibody        comprises a heavy chain variable region having at least 90%        sequence identity to an amino acid sequence selected from SEQ ID        NO:21-25 and a light chain variable region having at least 90%        sequence identity to an amino acid sequence selected from SEQ ID        NO:26-31. In one embodiment, the antibody comprises a heavy        chain variable region having at least 90% sequence identity to        the amino acid sequence of SEQ ID NO:24 and a light chain        variable region having at least 90% sequence identity to the        amino acid sequence of SEQ ID NO:29. In one embodiment, the        antibody comprises a heavy chain variable region comprising the        amino acid sequence of SEQ ID NO:24 and a light chain variable        region comprising the amino acid sequence of SEQ ID NO:29. In        one embodiment, the antibody comprises a heavy chain variable        region having at least 90% sequence identity to the amino acid        sequence of SEQ ID NO:25 and a light chain variable region        having at least 90% sequence identity to the amino acid sequence        of SEQ ID NO:30. In one embodiment, the antibody comprises a        heavy chain variable region comprising the amino acid sequence        of SEQ ID NO:25 and a light chain variable region comprising the        amino acid sequence of SEQ ID NO:30.

The following embodiments are further provided for any of the aboveimmunoconjugates. In one embodiment, an immunoconjugate has in vitro orin vivo cell killing activity. In one embodiment, the linker is attachedto the antibody through a thiol group on the antibody. In oneembodiment, the linker is cleavable by a protease. In one embodiment,the linker comprises a val-cit dipeptide. In one embodiment, the linkercomprises a p-aminobenzyl unit. In one embodiment, the p-aminobenzylunit is disposed between the drug and a protease cleavage site in thelinker. In one embodiment, the p-aminobenzyl unit isp-aminobenzyloxycarbonyl (PAB). In one embodiment, the linker comprises6-maleimidocaproyl. In one embodiment, the 6-maleimidocaproyl isdisposed between the antibody and a protease cleavage site in thelinker. The above embodiments may occur singly or in any combinationwith one another.

In one embodiment, the drug is selected from MMAE and MMAF. In oneembodiment, the immunoconjugate has the formula

wherein Ab is any of the above anti-TAT226 antibodies, S is a sulfuratom, and p ranges from 2 to 5. In one embodiment, the immunoconjugatehas the formula

wherein Ab is any of the above anti-TAT226 antibodies, S is a sulfuratom, and p ranges from 2 to 5.

In one aspect, a pharmaceutical composition is provided comprising anyof the above immunoconjugates and a pharmaceutically acceptable carrier.In one aspect, a method of treating a cell proliferative disorder isprovided, wherein the method comprises administering to an individualthe pharmaceutical composition. In one embodiment, the cellproliferative disorder is selected from ovarian cancer, uterine cancer,brain tumor, and Wilms' tumor. In one embodiment, the cell proliferativedisorder is associated with increased expression of TAT226 on thesurface of a cell.

In one aspect, a method of inhibiting cell proliferation is provided,wherein the method comprises exposing a cell to any of the aboveimmunoconjugates under conditions permissive for binding of theimmunoconjugate to TAT226. In one embodiment, the cell is a tumor cell.In one embodiment, the tumor cell is an ovarian tumor cell, a uterinetumor cell, a brain tumor cell, or a Wilms' tumor cell. In oneembodiment, the cell is a xenograft. In one embodiment, the exposingtakes place in vitro. In one embodiment, the exposing takes place invivo.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an alignment of TAT226 from human, cynomolgus monkey(“cyno”), mouse, and rat. Shaded residues are identical among thespecies. Unshaded residues differ between at least two of the fourspecies. Percent amino acid identity among the TAT226 sequences fromhuman, cynomolgus monkey, mouse, and rat is shown in the tableunderneath the alignment. Percent identity was calculated using theClustalW program.

FIG. 2 shows the H1, H2, and H3 heavy chain hypervariable region (HVR)sequences of anti-TAT226 monoclonal antibodies designated YWO.32 andYWO.49, as described in Example B Amino acid positions are numberedaccording to the Kabat numbering system as described below.

FIG. 3 shows the L1, L2, and L3 light chain HVR sequences of anti-TAT226monoclonal antibodies designated YWO.32 and YWO.49, as described inExample B. Amino acid positions are numbered according to the Kabatnumbering system as described below.

FIG. 4 shows the HVR-H3 and HVR-L3 sequences of YWO.49 and YWO.49.B7,YWO.49.C9, YWO.49.H2, and YWO.49.H6, which were generated by affinitymaturation of YWO.49 using HVR-H3 and HVR-L3 soft-randomized libraries,as described in Example B. Consensus HVR-H3 and HVR-L3 sequences arealso shown.

FIGS. 5A and 5B show exemplary acceptor human variable heavy (VH)consensus framework sequences for use in practicing the instantinvention with sequence identifiers as follows:

-   -   human VH subgroup I consensus framework “A” minus Kabat CDRs        (SEQ ID NOs:32, 33, 34, 35).    -   human VH subgroup I consensus frameworks “B,” “C,” and “D” minus        extended hypervariable regions (SEQ ID NOs:36, 37, 34, 35; SEQ        ID NOs:36, 37, 38, 35; and SEQ ID NOs:36, 37, 39, 35).    -   human VH subgroup II consensus framework “A” minus Kabat CDRs        (SEQ ID NOs:40, 41, 42, 35).    -   human VH subgroup II consensus frameworks “B,” “C,” and “D”        minus extended hypervariable regions (SEQ ID NOs:43, 44, 42, 35;        SEQ ID NOs:43, 44, 45, 35; and SEQ ID NOs:43, 44, 46, and 35).    -   human VH subgroup III consensus framework “A” minus Kabat CDRs        (SEQ ID NOs:47, 48, 49, 35).    -   human VH subgroup III consensus frameworks “B,” “C,” and “D”        minus extended hypervariable regions (SEQ ID NOs:50, 51, 49, 35;        SEQ ID NOs:50, 51, 52, 35; and SEQ ID NOs:50, 51, 53, 35).    -   human VH acceptor framework “A” minus Kabat CDRs (SEQ ID NOs:54,        48, 55, 35).    -   human VH acceptor frameworks “B” and “C” minus extended        hypervariable regions (SEQ ID NOs:50, 51, 55, 35; and SEQ ID        NOs:50, 51, 56, 35).    -   human VH acceptor 2 framework “A” minus Kabat CDRs (SEQ ID        NOs:54, 48, 57, 35).    -   human VH acceptor 2 framework “B,” “C,” and “D” minus extended        hypervariable regions (SEQ ID NOs:50, 51, 57, 35; SEQ ID NOs:50,        51, 58, 35; and SEQ ID NOs:50, 51, 59, 35).

FIGS. 6A and 6B show exemplary acceptor human variable light (VL)consensus framework sequences for use in practicing the instantinvention with sequence identifiers as follows:

-   -   human VL kappa subgroup I consensus framework (κv1): SEQ ID        NOs:60, 61, 62, 63    -   human VL kappa subgroup II consensus framework (κv2): SEQ ID        NOs:64, 65, 66, 63    -   human VL kappa subgroup III consensus framework (κv3): SEQ ID        NOs:67, 68, 69, 63    -   human VL kappa subgroup IV consensus framework (κv4): SEQ ID        NOs:70, 71, 72, 63

FIG. 7 shows framework sequences of huMAb4D5-8 light and heavy chains.Numbers in superscript/bold indicate amino acid positions according toKabat.

FIG. 8 shows framework sequences of huMAb4D5-8 light and heavy chainswith the indicated modifications. Numbers in superscript/bold indicateamino acid positions according to Kabat.

FIG. 9 shows the heavy chain variable region (VH) sequences of YWO.32,YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2, and YWO.49.H6. HVRs areunderlined.

FIG. 10 shows the light chain variable region (VL) sequences of YWO.32,YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2, and YWO.49.H6. VL sequences ofhumanized monoclonal antibody 4D5-8 (“huMAb4D5-8”) and a “modified”huMAb4D5-8 are also shown in SEQ ID NO:31 and SEQ ID NO:26,respectively. YWO.32 and YWO.49 have the same VL sequence as “modified”huMAb4D5-8 VL (SEQ ID NO:26), which contains the following substitutionsrelative to SEQ ID NO:31: N30S, R66G, and H91S. HVRs are underlined.

FIG. 11 shows an alignment of the heavy chain variable region sequencesof YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2, and YWO.49.H6. HVRsare enclosed in boxes. Residues of the HVR-H3s of YWO.49.B7, YWO.49.C9,YWO.49.H2, and YWO.49.H6 that differ from the corresponding residues ofthe HVR-H3 of YWO.49 are shaded.

FIG. 12 shows an alignment of the light chain variable region sequencesof YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2, and YWO.49.H6. HVRsare enclosed in boxes. Residues of the HVR-L3s of YWO.49.B7, YWO.49.C9,YWO.49.H2, and YWO.49.H6 that differ from the corresponding residues ofthe HVR-L3 of YWO.49 are shaded.

FIG. 13 shows a graphic representation of the levels of human TAT226gene expression in various tissues, as described in Example A.

FIG. 14 shows a graphic representation of the levels of human TAT226gene expression in normal ovary; normal fallopian tube; ovarian cancerof the clear cell, mucinous, and serous cystoadenocarcinoma subtypes;metastatic ovarian cancer; and other types of ovarian cancer, asdescribed in Example A.

FIG. 15 shows the results of fluorescence activated cell sorting (FACS)of OVCAR3 cells in the absence or presence of the indicated anti-TAT226antibodies, as described in Example D.

FIG. 16 shows TAT226 mRNA and protein expression as determined by a 5′nuclease (TaqMan®) assay and immunohistochemistry (IHC) performed onOVCAR3 cells and a panel of ovarian cancer samples, as described inExample F.

FIG. 17 shows the in vitro activity of various YWO.49.H2 and YWO.49.H6antibody-drug conjugates (ADCs) in an OVCAR3 cell killing assay, asdescribed in Example H.

FIG. 18 shows the in vitro activity of various YWO.49.H2 and YWO.49.H6ADCs in a cell killing assay using HCT116#9-4 stable transfectants, asdescribed in Example H.

FIG. 19 shows the in vivo activity of YWO.49.H6 ADCs using mousexenografts, as described in Example H.

FIG. 20 shows the in vivo activity of YWO.49.H6 ADCs using mousexenografts derived from human patient tumors, as described in Example H.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Isolated antibodies that bind to TAT226 are provided. Immunoconjugatescomprising anti-TAT226 antibodies are further provided. Antibodies andimmunoconjugates of the invention are useful, e.g., for the diagnosis ortreatment of disorders associated with altered expression, e.g.,increased expression, of TAT226. In certain embodiments, antibodies orimmunoconjugates of the invention are useful for the diagnosis ortreatment of a cell proliferative disorder, such as a tumor or cancer.In certain embodiments, antibodies or immunoconjugates of the inventionare useful for the detection of TAT226, e.g., TAT226 expressed on thecell surface.

Polynucleotides encoding anti-TAT226 antibodies are provided. Vectorscomprising polynucleotides encoding anti-TAT226 antibodies are provided,and host cells comprising such vectors are provided. Compositions,including pharmaceutical formulations, comprising any one or more of thepolynucleotides, anti-TAT226 antibodies, or immunoconjugates of theinvention are also provided.

I. GENERAL TECHNIQUES

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3rd. edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Current Protocols inMolecular Biology (F. M. Ausubel, et al. eds., (2003)); the seriesMethods in Enzymology (Academic Press, Inc.): Pcr 2: A PracticalApproach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and AnimalCell Culture (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; CellBiology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press;Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Celland Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press;Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B.Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbookof Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); GeneTransfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos,eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds.,1994); Current Protocols in Immunology (J. E. Coligan et al., eds.,1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999),Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRLPress, 1988-1989); Monoclonal Antibodies: A Practical Approach (P.Shepherd and C. Dean, eds., Oxford University Press, 2000); UsingAntibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold SpringHarbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D.Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principlesand Practice of Oncology (V. T. DeVita et al., eds., J.B. LippincottCompany, 1993).

II. DEFINITIONS AND ABBREVIATIONS

A. Definitions

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with research, diagnostic or therapeutic uses for theantibody, and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In some embodiments, an antibody is purified(1) to greater than 95% by weight of antibody as determined by, forexample, the Lowry method, and in some embodiments, to greater than 99%by weight; (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of, for example, aspinning cup sequenator, or (3) to homogeneity by SDS-PAGE underreducing or nonreducing conditions using, for example, Coomassie blue orsilver stain. Isolated antibody includes the antibody in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, isolated antibodywill be prepared by at least one purification step.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isseparated from at least one other nucleic acid molecule with which it isordinarily associated, for example, in its natural environment. Anisolated nucleic acid molecule further includes a nucleic acid moleculecontained in cells that ordinarily express the nucleic acid molecule,but the nucleic acid molecule is present extrachromosomally or at achromosomal location that is different from its natural chromosomallocation.

“Purified” means that a molecule is present in a sample at aconcentration of at least 95% by weight, or at least 98% by weight ofthe sample in which it is contained.

The term “substantially similar” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (for example, one associated with an antibody of theinvention and the other associated with a reference/comparatorantibody), such that one of skill in the art would consider thedifference between the two values to be of little or no biologicaland/or statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values). The differencebetween said two values is, for example, less than about 50%, less thanabout 40%, less than about 30%, less than about 20%, and/or less thanabout 10% as a function of the reference/comparator value.

The phrase “substantially reduced,” or “substantially different,” asused herein, denotes a sufficiently high degree of difference betweentwo numeric values (generally one associated with a molecule and theother associated with a reference/comparator molecule) such that one ofskill in the art would consider the difference between the two values tobe of statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values). The differencebetween said two values is, for example, greater than about 10%, greaterthan about 20%, greater than about 30%, greater than about 40%, and/orgreater than about 50% as a function of the value for thereference/comparator molecule.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA into which additional DNA segments may beligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors,” or simply, “expressionvectors.” In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase or by a syntheticreaction. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may comprise modification(s)made after synthesis, such as conjugation to a label. Other types ofmodifications include, for example, “caps,” substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotides(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars may be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or may be conjugated to solid or semi-solidsupports. The 5′ and 3′ terminal OH can be phosphorylated or substitutedwith amines or organic capping group moieties of from 1 to 20 carbonatoms. Other hydroxyls may also be derivatized to standard protectinggroups. Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl-, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such asarabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs, and basic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), (O)NR₂ (“amidate”), P(O)R,P(O)OR′, CO, or CH2 (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

“Oligonucleotide,” as used herein, generally refers to short, generallysingle-stranded, generally synthetic polynucleotides that are generally,but not necessarily, less than about 200 nucleotides in length. Theterms “oligonucleotide” and “polynucleotide” are not mutually exclusive.The description above for polynucleotides is equally and fullyapplicable to oligonucleotides.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “TAT226,” as used herein, refers to any native TAT226 from anyvertebrate source, including mammals such as primates (e.g. humans) androdents (e.g., mice and rats), unless otherwise indicated. The termencompasses “full-length,” unprocessed TAT226 as well as any form ofTAT226 that results from processing in the cell. The term alsoencompasses naturally occurring variants of TAT226, e.g., splicevariants or allelic variants. A “mature form” of TAT226 is a form ofTAT226 that has undergone processing, e.g., a form of TAT226 that hasundergone N-terminal (e.g., signal sequence) and/or C-terminal cleavageand/or modification by attachment of a GPI anchor. In one embodiment, a“mature form” of TAT226 is expressed on the cell surface.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingsimilar structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which generally lackantigen specificity. Polypeptides of the latter kind are, for example,produced at low levels by the lymph system and at increased levels bymyelomas.

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and include monoclonal antibodies (e.g., full lengthor intact monoclonal antibodies), polyclonal antibodies, monovalentantibodies, multivalent antibodies, multispecific antibodies (e.g.,bispecific antibodies so long as they exhibit the desired biologicalactivity) and may also include certain antibody fragments (as describedin greater detail herein). An antibody can be chimeric, human, humanizedand/or affinity matured.

The term “anti-TAT226 antibody” or “an antibody that binds to TAT226”refers to an antibody that is capable of binding TAT226 with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting TAT226. Preferably, the extent of bindingof an anti-TAT226 antibody to an unrelated, non-TAT226 protein is lessthan about 10% of the binding of the antibody to TAT226 as measured,e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibodythat binds to TAT226 has a dissociation constant (Kd) of ≦≦100 nM, ≦10nM, ≦1 nM, or ≦0.1 nM. In certain embodiments, an anti-TAT226 antibodybinds to an epitope of TAT226 that is conserved among TAT226 fromdifferent species.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domain of the heavy chain may be referred to as “VH.” Thevariable domain of the light chain may be referred to as “VL.” Thesedomains are generally the most variable parts of an antibody and containthe antigen-binding sites.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions (HVRs) both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework regions (FR). The variable domains of nativeheavy and light chains each comprise four FR regions, largely adopting abeta-sheet configuration, connected by three CDRs, which form loopsconnecting, and in some cases forming part of, the beta-sheet structure.The CDRs in each chain are held together in close proximity by the FRregions and, with the CDRs from the other chain, contribute to theformation of the antigen-binding site of antibodies (see Kabat et al.,Sequences of Proteins of Immunological Interest, Fifth Edition, NationalInstitute of Health, Bethesda, Md. (1991)). The constant domains are notinvolved directly in the binding of an antibody to an antigen, butexhibit various effector functions, such as participation of theantibody in antibody-dependent cellular toxicity.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequences of the constant domains of theirheavy chains, antibodies (immunoglobulins) can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavychain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known and described generally in, for example,Abbas et al. Cellular and Mol. Immunology, 4th ed. (2000). An antibodymay be part of a larger fusion molecule, formed by covalent ornon-covalent association of the antibody with one or more other proteinsor peptides.

The terms “full length antibody,” “intact antibody” and “whole antibody”are used herein interchangeably to refer to an antibody in itssubstantially intact form, not antibody fragments as defined below. Theterms particularly refer to an antibody with heavy chains that containthe Fc region.

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion retains at least one, and as many as most or all, ofthe functions normally associated with that portion when present in anintact antibody. In one embodiment, an antibody fragment comprises anantigen binding site of the intact antibody and thus retains the abilityto bind antigen. In another embodiment, an antibody fragment, forexample one that comprises the Fc region, retains at least one of thebiological functions normally associated with the Fc region when presentin an intact antibody, such as FcRn binding, antibody half lifemodulation, ADCC function and complement binding. In one embodiment, anantibody fragment is a monovalent antibody that has an in vivo half lifesubstantially similar to an intact antibody. For example, such anantibody fragment may comprise on antigen binding arm linked to an Fcsequence capable of conferring in vivo stability to the fragment.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-binding site. In one embodiment, a two-chain Fv species consistsof a dimer of one heavy- and one light-chain variable domain in tight,non-covalent association. In a single-chain Fv (scFv) species, oneheavy- and one light-chain variable domain can be covalently linked by aflexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment contains the heavy- and light-chain variable domainsand also contains the constant domain of the light chain and the firstconstant domain (CH1) of the heavy chain. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody hinge region. Fab′-SH is the designation herein for Fab′ inwhich the cysteine residue(s) of the constant domains bear a free thiolgroup. F(ab′)₂ antibody fragments originally were produced as pairs ofFab′ fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFvsee Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies may be bivalent orbispecific. Diabodies are described more fully in, for example, EP404,097; WO93/1161; Hudson et al. (2003) Nat. Med. 9:129-134; andHollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).Triabodies and tetrabodies are also described in Hudson et al. (2003)Nat. Med. 9:129-134.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible mutations, e.g., naturally occurring mutations, thatmay be present in minor amounts. Thus, the modifier “monoclonal”indicates the character of the antibody as not being a mixture ofdiscrete antibodies. In certain embodiments, such a monoclonal antibodytypically includes an antibody comprising a polypeptide sequence thatbinds a target, wherein the target-binding polypeptide sequence wasobtained by a process that includes the selection of a single targetbinding polypeptide sequence from a plurality of polypeptide sequences.For example, the selection process can be the selection of a uniqueclone from a plurality of clones, such as a pool of hybridoma clones,phage clones, or recombinant DNA clones. It should be understood that aselected target binding sequence can be further altered, for example, toimprove affinity for the target, to humanize the target bindingsequence, to improve its production in cell culture, to reduce itsimmunogenicity in vivo, to create a multispecific antibody, etc., andthat an antibody comprising the altered target binding sequence is alsoa monoclonal antibody of this invention. In contrast to polyclonalantibody preparations which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody of a monoclonal antibody preparation is directed against asingle determinant on an antigen. In addition to their specificity,monoclonal antibody preparations are advantageous in that they aretypically uncontaminated by other immunoglobulins.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by a variety of techniques, including, for example, the hybridomamethod (e.g., Kohler et al., Nature, 256: 495 (1975); Harlow et al.,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2^(nd) ed. 1988); Hammerling et al., in: Monoclonal Antibodies andT-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNAmethods (see, e.g., U.S. Pat. No. 4,816,567), phage display technologies(see, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al.,J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004);Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); andLee et al., J. Immunol. Methods 284(1-2): 119-132 (2004), andtechnologies for producing human or human-like antibodies in animalsthat have parts or all of the human immunoglobulin loci or genesencoding human immunoglobulin sequences (see, e.g., WO98/24893;WO96/34096; WO96/33735; WO91/10741; Jakobovits et al., Proc. Natl. Acad.Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993);Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; Markset al., Bio. Technology 10: 779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al.,Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996) and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93(1995).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit, or nonhuman primate having the desired specificity,affinity, and/or capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications may be made to further refine antibodyperformance. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin, and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the followingreview articles and references cited therein: Vaswani and Hamilton, Ann.Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc.Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.5:428-433 (1994).

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six hypervariable regions; three in the VH (H1, H2, H3), andthree in the VL (L1, L2, L3). In native antibodies, H3 and L3 displaythe most diversity of the six hypervariable regions, and H3 inparticular is believed to play a unique role in conferring finespecificity to antibodies. Xu et al. (2000) Immunity 13:37-45; Johnsonand Wu (2003) in Methods in Molecular Biology 248:1-25 (Lo, ed., HumanPress, Totowa, N.J.). Indeed, naturally occurring camelid antibodiesconsisting of a heavy chain only are functional and stable in theabsence of light chain. Hamers-Casterman et al. (1993) Nature363:446-448; Sheriff et al. (1996) Nature Struct. Biol. 3:733-736.

A number of hypervariable region delineations are in use and areencompassed herein. The Kabat Complementarity Determining Regions (CDRs)are based on sequence variability and are the most commonly used (Kabatet al., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991)).Chothia refers instead to the location of the structural loops (Chothiaand Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM hypervariableregions represent a compromise between the Kabat CDRs and Chothiastructural loops, and are used by Oxford Molecular's AbM antibodymodeling software. The “contact” hypervariable regions are based on ananalysis of the available complex crystal structures. The residues fromeach of these hypervariable regions are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

Hypervariable regions may comprise “extended hypervariable regions” asfollows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96(L3) in the VL and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102,or 95-102 (H3) in the VH. The variable domain residues are numberedaccording to Kabat et al., supra, for each of these definitions.

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues as herein defined.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat,” and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,Sequences of Proteins of Immunological Interest, 5^(th) Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991).Using this numbering system, the actual linear amino acid sequence maycontain fewer or additional amino acids corresponding to a shorteningof, or insertion into, a FR or HVR of the variable domain. For example,a heavy chain variable domain may include a single amino acid insert(residue 52a according to Kabat) after residue 52 of H2 and insertedresidues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat)after heavy chain FR residue 82. The Kabat numbering of residues may bedetermined for a given antibody by alignment at regions of homology ofthe sequence of the antibody with a “standard” Kabat numbered sequence.

An “affinity matured” antibody is one with one or more alterations inone or more HVRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). In one embodiment, an affinity maturedantibody has nanomolar or even picomolar affinities for the targetantigen. Affinity matured antibodies are produced by procedures known inthe art. Marks et al. Bio/Technology 10:779-783 (1992) describesaffinity maturation by VH and VL domain shuffling. Random mutagenesis ofHVR and/or framework residues is described by: Barbas et al. Proc Nat.Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155(1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al.,J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol.226:889-896 (1992).

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds. Certain blockingantibodies or antagonist antibodies substantially or completely inhibitthe biological activity of the antigen.

An “agonist antibody,” as used herein, is an antibody which mimics atleast one of the functional activities of a polypeptide of interest.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. In some embodiments, an FcR is a native human FcR. Insome embodiments, an FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof those receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see Daëron, Annu.Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch andKinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41(1995). Other FcRs, including those to be identified in the future, areencompassed by the term “FcR” herein.

The term “Fc receptor” or “FcR” also includes the neonatal receptor,FcRn, which is responsible for the transfer of maternal IgGs to thefetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J.Immunol. 24:249 (1994)) and regulation of homeostasis ofimmunoglobulins. Methods of measuring binding to FcRn are known (see,e.g., Ghetie 1997, Hinton 2004). Binding to human FcRn in vivo and serumhalf life of human FcRn high affinity binding polypeptides can beassayed, e.g., in transgenic mice or transfected human cell linesexpressing human FcRn, or in primates administered with the Fc variantpolypeptides.

WO00/42072 (Presta) describes antibody variants with improved ordiminished binding to FcRs. The content of that patent publication isspecifically incorporated herein by reference. See, also, Shields et al.J. Biol. Chem. 9(2): 6591-6604 (2001).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. In certain embodiments, the cells express atleast FcγRIII and perform ADCC effector function(s). Examples of humanleukocytes which mediate ADCC include peripheral blood mononuclear cells(PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells andneutrophils. The effector cells may be isolated from a native source,e.g., from blood.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g. Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The primary cells for mediatingADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI,FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarizedin Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92(1991). To assess ADCC activity of a molecule of interest, an in vitroADCC assay, such as that described in U.S. Pat. No. 5,500,362 or5,821,337 or Presta U.S. Pat. No. 6,737,056 may be performed. Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in a animal model such as that disclosed in Clynes et al.PNAS (USA) 95:652-656 (1998).

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996), may be performed.

Polypeptide variants with altered Fc region amino acid sequences andincreased or decreased C1q binding capability are described in U.S. Pat.No. 6,194,551B1 and WO99/51642. The contents of those patentpublications are specifically incorporated herein by reference. See,also, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

The term “Fc region-comprising polypeptide” refers to a polypeptide,such as an antibody or immunoadhesin, which comprises an Fc region. TheC-terminal lysine (residue 447 according to the EU numbering system) ofthe Fc region may be removed, for example, during purification of thepolypeptide or by recombinant engineering the nucleic acid encoding thepolypeptide. Accordingly, a composition comprising a polypeptide havingan Fc region according to this invention can comprise polypeptides withK447, with all K447 removed, or a mixture of polypeptides with andwithout the K447 residue.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a VL or VH framework derived froma human immunoglobulin framework or a human consensus framework. Anacceptor human framework “derived from” a human immunoglobulin frameworkor a human consensus framework may comprise the same amino acid sequencethereof, or it may contain pre-existing amino acid sequence changes. Insome embodiments, the number of pre-existing amino acid changes are 10or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 orless, 3 or less, or 2 or less. Where pre-existing amino acid changes arepresent in a VH, preferably those changes occur at only three, two, orone of positions 71H, 73H and 78H; for instance, the amino acid residuesat those positions may be 71A, 73T and/or 78A. In one embodiment, the VLacceptor human framework is identical in sequence to the VL humanimmunoglobulin framework sequence or human consensus framework sequence.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, 5^(th)Ed. Public Health Service, National Institutes of Health, Bethesda, Md.(1991). In one embodiment, for the VL, the subgroup is subgroup kappa Ias in Kabat et al., supra. In one embodiment, for the VH, the subgroupis subgroup III as in Kabat et al., supra.

A “VH subgroup III consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable heavy subgroup III ofKabat et al., supra. In one embodiment, the VH subgroup III consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ IDNO:50)-H1-WVRQAPGKGLEWV (SEQ ID NO:51)-H2-RFTISADTSKNTAYLQMNSLRAEDTAVYYC(SEQ ID NO:59)-H3-WGQGTLVTVSS (SEQ ID NO:35).

A “VL subgroup I consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable light kappa subgroupI of Kabat et al., supra. In one embodiment, the VH subgroup I consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences: DIQMTQSPSSLSASVGDRVTITC (SEQ IDNO:60)-L1-WYQQKPGKAPKLLIY (SEQ IDNO:61)-L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:62)-L3-FGQGTKVEIK(SEQ ID NO:63).

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention. Specific illustrative embodiments are describedin the following.

In one embodiment, the “Kd” or “Kd value” according to this invention ismeasured by a radiolabeled antigen binding assay (RIA) performed withthe Fab version of an antibody of interest and its antigen as describedby the following assay. Solution binding affinity of Fabs for antigen ismeasured by equilibrating Fab with a minimal concentration of(¹²⁵I)-labeled antigen in the presence of a titration series ofunlabeled antigen, then capturing bound antigen with an anti-Fabantibody-coated plate (Chen, et al., (1999) J. Mol. Biol. 293:865-881).To establish conditions for the assay, microtiter plates (Dynex) arecoated overnight with 5 μg/ml of a capturing anti-Fab antibody (CappelLabs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with2% (w/v) bovine serum albumin in PBS for two to five hours at roomtemperature (approximately 23° C.). In a non-adsorbent plate (Nunc#269620), 100 pM or 26 pM [¹²⁵I]-antigen are mixed with serial dilutionsof a Fab of interest (e.g., consistent with assessment of the anti-VEGFantibody, Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599).The Fab of interest is then incubated overnight; however, the incubationmay continue for a longer period (e.g., about 65 hours) to ensure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation at room temperature (e.g., for one hour).The solution is then removed and the plate washed eight times with 0.1%Tween-20 in PBS. When the plates have dried, 150 μl/well of scintillant(MicroScint-20; Packard) is added, and the plates are counted on aTopcount gamma counter (Packard) for ten minutes. Concentrations of eachFab that give less than or equal to 20% of maximal binding are chosenfor use in competitive binding assays.

According to another embodiment, the Kd or Kd value is measured by usingsurface plasmon resonance assays using a BIAcore™-2000 or aBIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. withimmobilized antigen CM5 chips at ˜10 response units (RU). Briefly,carboxymethylated dextran biosensor chips (CMS, BIAcore Inc.) areactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% Tween 20 (PBST) at 25° C. at a flow rate of approximately 25μl/min. Association rates (k_(on)) and dissociation rates (k_(off)) arecalculated using a simple one-to-one Langmuir binding model (BIAcoreEvaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (Kd) is calculated as the ratio k_(off)/k_(on). See, e.g.,Chen, Y., et al., (1999) J. Mol. Biol. 293:865-881. If the on-rateexceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmon resonance assay above, thenthe on-rate can be determined by using a fluorescent quenching techniquethat measures the increase or decrease in fluorescence emissionintensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25°C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in thepresence of increasing concentrations of antigen as measured in aspectrometer, such as a stop-flow equipped spectrophometer (AvivInstruments) or a 8000-series SLM-Aminco spectrophotometer(ThermoSpectronic) with a stirred cuvette.

An “on-rate,” “rate of association,” “association rate,” or “k_(on)”according to this invention can also be determined as described aboveusing a BIAcore™-2000 or a BIAcore™-3000 system (BIAcore, Inc.,Piscataway, N.J.).

A “disorder” is any condition or disease that would benefit fromtreatment with an substance/molecule or method of the invention. Thisincludes chronic and acute disorders including those pathologicalconditions which predispose the mammal to the disorder in question.Non-limiting examples of disorders to be treated herein includecancerous conditions such as tumors, e.g., carcinomas (epithelialtumors) and blastomas (embryonic tissue-derived tumors), and in someembodiments, ovarian cancer, uterine cancer (including endometrialcancer), brain tumors (e.g., astrocytomas and gliomas), and kidneycancer, including nephroblastomas (e.g., Wilms' tumor).

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer.

“Tumor,” as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. The terms “cancer,” “cancerous,” “cellproliferative disorder,” “proliferative disorder” and “tumor” are notmutually exclusive as referred to herein.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include, butare not limited to, carcinoma, lymphoma (e.g., Hodgkin's andnon-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer,hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, leukemia and other lymphoproliferative disorders, and varioustypes of head and neck cancer.

As used herein, “treatment” (and variations such as “treat” or“treating”) refers to clinical intervention in an attempt to alter thenatural course of the individual or cell being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include preventing occurrenceor recurrence of disease, alleviation of symptoms, diminishment of anydirect or indirect pathological consequences of the disease, preventingmetastasis, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis. Insome embodiments, antibodies of the invention are used to delaydevelopment of a disease or disorder or to slow the progression of adisease or disorder.

An “individual” is a vertebrate. In certain embodiments, the vertebrateis a mammal. Mammals include, but are not limited to, farm animals (suchas cows), sport animals, pets (such as cats, dogs, and horses),primates, mice and rats. In certain embodiments, a mammal is a human.

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic orprophylactic result.

A “therapeutically effective amount” of a substance/molecule of theinvention may vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of thesubstance/molecule, to elicit a desired response in the individual. Atherapeutically effective amount encompasses an amount in which anytoxic or detrimental effects of the substance/molecule are outweighed bythe therapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,but not necessarily, since a prophylactic dose is used in subjects priorto or at an earlier stage of disease, the prophylactically effectiveamount would be less than the therapeutically effective amount.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. The term is intended to include radioactive isotopes (e.g.,At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² andradioactive isotopes of Lu), chemotherapeutic agents (e.g.,methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine,etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil,daunorubicin or other intercalating agents, enzymes and fragmentsthereof such as nucleolytic enzymes, antibiotics, and toxins such assmall molecule toxins or enzymatically active toxins of bacterial,fungal, plant or animal origin, including fragments and/or variantsthereof, and the various antitumor or anticancer agents disclosed below.Other cytotoxic agents are described below. A tumoricidal agent causesdestruction of tumor cells.

A “toxin” is any substance capable of having a detrimental effect on thegrowth or proliferation of a cell.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,triethylenephosphoramide, triethylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®);beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin(including the synthetic analogue topotecan (HYCAMTIN®), CPT-11(irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including itsadozelesin, carzelesin and bizelesin synthetic analogues);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosoureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g.,Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antiobiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, caminomycin, carzinophilin, chromomycins,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, porfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoids, e.g., TAXOL® paclitaxel(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® docetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine(VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine(NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine (XELODA®);pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovovin.

Also included in this definition are anti-hormonal agents that act toregulate, reduce, block, or inhibit the effects of hormones that canpromote the growth of cancer, and are often in the form of systemic, orwhole-body treatment. They may be hormones themselves. Examples includeanti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including NOLVADEX® tamoxifen),EVISTA® raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY117018, onapristone, and FARESTON® toremifene;anti-progesterones; estrogen receptor down-regulators (ERDs); agentsthat function to suppress or shut down the ovaries, for example,leutinizing hormone-releasing hormone (LHRH) agonists such as LUPRON®and ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetateand tripterelin; other anti-androgens such as flutamide, nilutamide andbicalutamide; and aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole. Inaddition, such definition of chemotherapeutic agents includesbisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®),DIDROCAL® etidronate, NE-58095, ZOMETA® zoledronic acid/zoledronate,FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate, orACTONEL® risedronate; as well as troxacitabine (a 1,3-dioxolanenucleoside cytosine analog); antisense oligonucleotides, particularlythose that inhibit expression of genes in signaling pathways implicatedin abherant cell proliferation, such as, for example, PKC-alpha, Raf,H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such asTHERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN®vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; LURTOTECAN®topoisomerase 1 inhibitor; ABARELIX® rmRH; lapatinib ditosylate (anErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also knownas GW572016); and pharmaceutically acceptable salts, acids orderivatives of any of the above.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell (such as a cell expressingTAT226) either in vitro or in vivo. Thus, the growth inhibitory agentmay be one which significantly reduces the percentage of cells (such asa cell expressing TAT226) in S phase. Examples of growth inhibitoryagents include agents that block cell cycle progression (at a placeother than S phase), such as agents that induce G1 arrest and M-phasearrest. Classical M-phase blockers include the vincas (vincristine andvinblastine), taxanes, and topoisomerase II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Thoseagents that arrest G1 also spill over into S-phase arrest, for example,DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders:Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel anddocetaxel) are anticancer drugs both derived from the yew tree.Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the Europeanyew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-MyersSquibb). Paclitaxel and docetaxel promote the assembly of microtubulesfrom tubulin dimers and stabilize microtubules by preventingdepolymerization, which results in the inhibition of mitosis in cells.

The term “intracellular metabolite” refers to a compound resulting froma metabolic process or reaction inside a cell on an antibody-drugconjugate (ADC). The metabolic process or reaction may be an enzymaticprocess, such as proteolytic cleavage of a peptide linker of the ADC, orhydrolysis of a functional group such as a hydrazone, ester, or amide.Intracellular metabolites include, but are not limited to, antibodiesand free drug which have undergone intracellular cleavage after entry,diffusion, uptake or transport into a cell.

The terms “intracellularly cleaved” and “intracellular cleavage” referto a metabolic process or reaction inside a cell on an antibody-drugconjugate (ADC) whereby the covalent attachment, i.e. linker, betweenthe drug moiety (D) and the antibody (Ab) is broken, resulting in thefree drug dissociated from the antibody inside the cell. The cleavedmoieties of the ADC are thus intracellular metabolites.

The term “bioavailability” refers to the systemic availability (i.e.,blood/plasma levels) of a given amount of drug administered to apatient. Bioavailability is an absolute term that indicates measurementof both the time (rate) and total amount (extent) of drug that reachesthe general circulation from an administered dosage form.

The term “cytotoxic activity” refers to a cell-killing, cytostatic orgrowth inhibitory effect of an antibody-drug conjugate or anintracellular metabolite of an antibody-drug conjugate. Cytotoxicactivity may be expressed as the IC₅₀ value, which is the concentration(molar or mass) per unit volume at which half the cells survive.

“Alkyl” is C₁-C₁₈ hydrocarbon containing normal, secondary, tertiary orcyclic carbon atoms. Examples are methyl (Me, —CH₃), ethyl (Et,—CH₂CH₃), 1-propyl (n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr,i-propyl, —CH(CH₃)₂), 1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃),2-methyl-1-propyl (i-Bu, i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl,—CH(CH₃)CH₂CH₃), 2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl(n-pentyl, —CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃.

The term “C₁-C₈ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 8 carbonatoms. Representative “C₁-C₈ alkyl” groups include, but are not limitedto, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl,-n-heptyl, -n-octyl, -n-nonyl and -n-decyl; while branched C₁-C₈ alkylsinclude, but are not limited to, -isopropyl, -sec-butyl, -isobutyl,-tert-butyl, -isopentyl, 2-methylbutyl, unsaturated C₁-C₈ alkylsinclude, but are not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl,-isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl,-2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl,3-hexylacetylenyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl,-2-pentynyl, -3-methyl-1 butynyl, methyl, ethyl, propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl,neopentyl, n-hexyl, isohexyl, 2-methylpentyl, 3-methylpentyl,2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl,2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl,3-methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl,3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl,n-heptyl, isoheptyl, n-octyl, and isooctyl. A C₁-C₈ alkyl group can beunsubstituted or substituted with one or more groups including, but notlimited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —SO₃R′, —S(O)₂R′,—S(O)R′, —OH, -halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where eachR′ is independently selected from H, —C₁-C₈ alkyl and aryl.

“Alkenyl” is C₂-C₁₈ hydrocarbon containing normal, secondary, tertiaryor cyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp² double bond. Examples include, but are not limitedto: ethylene or vinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), cyclopentenyl(—C₅H₇), and 5-hexenyl (—CH₂ CH₂CH₂CH₂CH═CH₂)

“Alkynyl” is C₂-C₁₈ hydrocarbon containing normal, secondary, tertiaryor cyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp triple bond. Examples include, but are not limited to:acetylenic (—C≡CH) and propargyl (—CH₂C≡CH),

“Alkylene” refers to a saturated, branched or straight chain or cyclichydrocarbon radical of 1-18 carbon atoms, and having two monovalentradical centers derived by the removal of two hydrogen atoms from thesame or two different carbon atoms of a parent alkane. Typical alkyleneradicals include, but are not limited to: methylene (—CH₂—) 1,2-ethyl(—CH₂CH₂—), 1,3-propyl (—CH₂CH₂CH₂—), 1,4-butyl (—CH₂CH₂CH₂CH₂—), andthe like.

A “C₁-C₁₀ alkylene” is a straight chain, saturated hydrocarbon group ofthe formula —(CH₂)₁₋₁₀—. Examples of a C₁-C₁₀ alkylene includemethylene, ethylene, propylene, butylene, pentylene, hexylene,heptylene, ocytylene, nonylene and decalene.

“Alkenylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkene. Typicalalkenylene radicals include, but are not limited to: 1,2-ethylene(—CH═CH—).

“Alkynylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkyne. Typicalalkynylene radicals include, but are not limited to: acetylene (—C≡C—),propargyl (—CH₂C≡C—), and 4-pentynyl (—CH₂CH₂CH₂C≡C—).

“Aryl” refers to a carbocyclic aromatic group. Examples of aryl groupsinclude, but are not limited to, phenyl, naphthyl and anthracenyl. Acarbocyclic aromatic group or a heterocyclic aromatic group can beunsubstituted or substituted with one or more groups including, but notlimited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′,—OH, -halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

An “arylene” is an aryl group which has two covalent bonds and can be inthe ortho, meta, or para configurations as shown in the followingstructures:

in which the phenyl group can be unsubstituted or substituted with up tofour groups including, but not limited to, —C₁-C₈ alkyl, —O—(C₁-C₈alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃, —NH₂,—NH(R′), —N(R′)₂ and —CN; wherein each R′ is independently selected fromH, —C₁-C₈ alkyl and aryl.

“Arylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with an aryl radical. Typical arylalkyl groupsinclude, but are not limited to, benzyl, 2-phenylethan-1-yl,2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and thelike. The arylalkyl group comprises 6 to 20 carbon atoms, e.g. the alkylmoiety, including alkanyl, alkenyl or alkynyl groups, of the arylalkylgroup is 1 to 6 carbon atoms and the aryl moiety is 5 to 14 carbonatoms.

“Heteroarylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with a heteroaryl radical. Typicalheteroarylalkyl groups include, but are not limited to,2-benzimidazolylmethyl, 2-furylethyl, and the like. The heteroarylalkylgroup comprises 6 to 20 carbon atoms, e.g. the alkyl moiety, includingalkanyl, alkenyl or alkynyl groups, of the heteroarylalkyl group is 1 to6 carbon atoms and the heteroaryl moiety is 5 to 14 carbon atoms and 1to 3 heteroatoms selected from N, O, P, and S. The heteroaryl moiety ofthe heteroarylalkyl group may be a monocycle having 3 to 7 ring members(2 to 6 carbon atoms or a bicycle having 7 to 10 ring members (4 to 9carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), forexample: a bicyclo [4,5], [5,5], [5,6], or [6,6] system.

“Substituted alkyl,” “substituted aryl,” and “substituted arylalkyl”mean alkyl, aryl, and arylalkyl respectively, in which one or morehydrogen atoms are each independently replaced with a substituent.Typical substituents include, but are not limited to, —X, —R, —O⁻, —OR,—SR, —S⁻, —NR₂, —NR₃, ═NR, —CX₃, —CN, —OCN, —SCN, —N═C═O, —NCS, —NO,—NO₂, ═N₂, —N₃, NC(═O)R, —C(═O)R, —C(═O)NR₂, —SO₃ ⁻, —SO₃H, —S(═O)₂R,—OS(═O)₂OR, —S(═O)₂NR, —S(═O)R, —OP(═O)(OR)₂, —P(═O)(OR)₂, —PO₃H₂,—C(═O)R, —C(═O)X, —C(═S)R, —CO₂R, —CO₂ ⁻, —C(═S)OR, —C(═O)SR, —C(═S)SR,—C(═O)NR₂, —C(═S)NR₂, —C(═NR)NR₂, where each X is independently ahalogen: F, Cl, Br, or I; and each R is independently —H, C₂-C₁₈ alkyl,C₆-C₂₀ aryl, C₃-C₁₄ heterocycle, protecting group or prodrug moiety.Alkylene, alkenylene, and alkynylene groups as described above may alsobe similarly substituted.

“Heteroaryl” and “heterocycle” refer to a ring system in which one ormore ring atoms is a heteroatom, e.g. nitrogen, oxygen, and sulfur. Theheterocycle radical comprises 1 to 20 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S. A heterocycle may be amonocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S) or a bicycle having 7 to 10ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected fromN, O, P, and S), for example: a bicyclo [4,5], [5,5], [5,6], or [6,6]system.

Heterocycles are described in Paquette, Leo A.; “Principles of ModernHeterocyclic Chemistry” (W. A. Benjamin, New York, 1968), particularlyChapters 1, 3, 4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds,A series of Monographs” (John Wiley & Sons, New York, 1950 to present),in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc.(1960) 82:5566.

Examples of heterocycles include by way of example and not limitationpyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl,tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl,furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl,benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl,isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl,2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl,tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl,azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl,thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl,phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl,pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl,4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl,β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl,chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl,oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,and isatinoyl.

By way of example and not limitation, carbon bonded heterocycles arebonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2,3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline. Still more typically, carbon bonded heterocycles include2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl,4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl,4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl,5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

By way of example and not limitation, nitrogen bonded heterocycles arebonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine,2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline,3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline,piperidine, piperazine, indole, indoline, 1H-indazole, position 2 of aisoindole, or isoindoline, position 4 of a morpholine, and position 9 ofa carbazole, or β-carboline. Still more typically, nitrogen bondedheterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl,1-pyrazolyl, and 1-piperidinyl.

A “C₃-C₈ heterocycle” refers to an aromatic or non-aromatic C₃-C₈carbocycle in which one to four of the ring carbon atoms areindependently replaced with a heteroatom from the group consisting of O,S and N. Representative examples of a C₃-C₈ heterocycle include, but arenot limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl,coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl,imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl,pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl andtetrazolyl. A C₃-C₈ heterocycle can be unsubstituted or substituted withup to seven groups including, but not limited to, —C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃,—NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ is independentlyselected from H, —C₁-C₈ alkyl and aryl.

“C₃-C₈ heterocyclo” refers to a C₃-C₈ heterocycle group defined abovewherein one of the heterocycle group's hydrogen atoms is replaced with abond. A C₃-C₈ heterocyclo can be unsubstituted or substituted with up tosix groups including, but not limited to, —C₁-C₈ alkyl, —O—(C₁-C₈alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃, —NH₂,—NH(R′), —N(R′)₂ and —CN; wherein each R′ is independently selected fromH, —C₁-C₈ alkyl and aryl.

“Carbocycle” means a saturated or unsaturated ring having 3 to 7 carbonatoms as a monocycle or 7 to 12 carbon atoms as a bicycle. Monocycliccarbocycles have 3 to 6 ring atoms, still more typically 5 or 6 ringatoms. Bicyclic carbocycles have 7 to 12 ring atoms, e.g. arranged as abicyclo [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atomsarranged as a bicyclo [5,6] or [6,6] system. Examples of monocycliccarbocycles include cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl,and cyclooctyl.

A “C₃-C₈ carbocycle” is a 3-, 4-, 5-, 6-, 7- or 8-membered saturated orunsaturated non-aromatic carbocyclic ring. Representative C₃-C₈carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl,-cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl,-1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl,-1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and-cyclooctadienyl. A C₃-C₈ carbocycle group can be unsubstituted orsubstituted with one or more groups including, but not limited to,—C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′,—C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH,-halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

A “C₃-C₈ carbocyclo” refers to a C₃-C₈ carbocycle group defined abovewherein one of the carbocycle groups' hydrogen atoms is replaced with abond.

“Linker” refers to a chemical moiety comprising a covalent bond or achain of atoms that covalently attaches an antibody to a drug moiety. Invarious embodiments, linkers include a divalent radical such as analkyldiyl, an aryldiyl, a heteroaryldiyl, moieties such as:—(CR₂)_(n)O(CR₂)_(n)—, repeating units of alkyloxy (e.g. polyethylenoxy,PEG, polymethyleneoxy) and alkylamino (e.g. polyethyleneamino,Jeffamine™); and diacid ester and amides including succinate,succinamide, diglycolate, malonate, and caproamide.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., NewYork. Many organic compounds exist in optically active forms, i.e., theyhave the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L, or R andS, are used to denote the absolute configuration of the molecule aboutits chiral center(s). The prefixes d and l or (+) and (−) are employedto designate the sign of rotation of plane-polarized light by thecompound, with (−) or 1 meaning that the compound is levorotatory. Acompound prefixed with (+) or d is dextrorotatory. For a given chemicalstructure, these stereoisomers are identical except that they are mirrorimages of one another. A specific stereoisomer may also be referred toas an enantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity.

“Leaving group” refers to a functional group that can be substituted byanother functional group. Certain leaving groups are well known in theart, and examples include, but are not limited to, a halide (e.g.,chloride, bromide, iodide), methanesulfonyl (mesyl), p-toluenesulfonyl(tosyl), trifluoromethylsulfonyl (triflate), andtrifluoromethylsulfonate.

B. Abbreviations

Linker Components:

-   -   MC=6-maleimidocaproyl    -   Val-Cit or “vc”=valine-citrulline (an exemplary dipeptide in a        protease cleavable linker)    -   Citrulline=2-amino-5-ureido pentanoic acid    -   PAB=p-aminobenzyloxycarbonyl (an example of a “self immolative”        linker component)    -   Me-Val-Cit=N-methyl-valine-citrulline (wherein the linker        peptide bond has been modified to prevent its cleavage by        cathepsin B)    -   MC(PEG)6-OH=maleimidocaproyl-polyethylene glycol (can be        attached to antibody cysteines).

Cytotoxic Drugs:

-   -   MMAE=mono-methyl auristatin E (MW 718)    -   MMAF=variant of auristatin E (MMAE) with a phenylalanine at the        C-terminus of the drug (MW 731.5)    -   MMAF-DMAEA=MMAF with DMAEA (dimethylaminoethylamine) in an amide        linkage to the C-terminal phenylalanine (MW 801.5)    -   MMAF-TEG=MMAF with tetraethylene glycol esterified to the        phenylalanine    -   MMAF-NtBu=N-t-butyl, attached as an amide to C-terminus of MMAF

Further abbreviations are as follows: AE is auristatin E, Boc isN-(t-butoxycarbonyl), cit is citrulline, dap is dolaproine, DCC is1,3-dicyclohexylcarbodiimide, DCM is dichloromethane, DEA isdiethylamine, DEAD is diethylazodicarboxylate, DEPC isdiethylphosphorylcyanidate, DIAD is diisopropylazodicarboxylate, DIEA isN,N-diisopropylethylamine, dil is dolaisoleucine, DMA isdimethylacetamide, DMAP is 4-dimethylaminopyridine, DME isethyleneglycol dimethyl ether (or 1,2-dimethoxyethane), DMF isN,N-dimethylformamide, DMSO is dimethylsulfoxide, doe is dolaphenine,dov is N,N-dimethylvaline, DTNB is 5,5′-dithiobis(2-nitrobenzoic acid),DTPA is diethylenetriaminepentaacetic acid, DTT is dithiothreitol, EDCIis 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EEDQ is2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, ES-MS is electrospraymass spectrometry, EtOAc is ethyl acetate, Fmoc isN-(9-fluorenylmethoxycarbonyl), gly is glycine, HATU isO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate, HOBt is 1-hydroxybenzotriazole, HPLC is highpressure liquid chromatography, ile is isoleucine, lys is lysine,MeCN(CH₃CN) is acetonitrile, MeOH is methanol, Mtr is4-anisyldiphenylmethyl (or 4-methoxytrityl), nor is(1S,2R)-(+)-norephedrine, PBS is phosphate-buffered saline (pH 7.4), PEGis polyethylene glycol, Ph is phenyl, Pnp is p-nitrophenyl, MC is6-maleimidocaproyl, phe is L-phenylalanine, PyBrop is bromotris-pyrrolidino phosphonium hexafluorophosphate, SEC is size-exclusionchromatography, Su is succinimide, TFA is trifluoroacetic acid, TLC isthin layer chromatography, UV is ultraviolet, and val is valine.

III. COMPOSITIONS AND METHODS OF MAKING THE SAME

Antibodies that bind to TAT226 are provided. Immunoconjugates comprisinganti-TAT226 antibodies are provided. Antibodies and immunoconjugates ofthe invention are useful, e.g., for the diagnosis or treatment ofdisorders associated with altered expression, e.g., increasedexpression, of TAT226. In certain embodiments, antibodies orimmunoconjugates of the invention are useful for the diagnosis ortreatment of a cell proliferative disorder, such as cancer.

A. Anti-TAT226 Antibodies

TAT226 (“Tumor-associated Antigenic Target no. 226”) is a protein thatis processed and expressed on the surface of certain cell types,including tumor cells. In particular, human TAT226 has previously beenreported to be overexpressed in certain types of tumors, includingovarian, uterine, endometrial, kidney, lung, pancreatic, adrenal, andhepatocellular tumors. See, e.g., U.S. Patent Application PublicationNos. US 2003/0148408 A1, US 2004/0229277 A1, and US 2003/0100712 A1(referring to TAT226 as “PRO9917”); and U.S. Pat. No. 6,710,170 B2 (SEQID NO:215). Other database entries and disclosures related to TAT226 areas follows: NCBI accession no. AY3586281 (referring to human TAT226 as“PSCA Hlog”); NCBI accession no. AAQ88991.1 and “gi” no. 37182378(referring to human TAT226 as “PSCA Hlog”); RIKEN cDNA 2700050C12; US2003/0096961 A1 (SEQ ID NO:16); US 2003/0129192 A1 (SEQ ID NO:215); US2003/0206918 A1 (Example 5; SEQ ID NO:215); US 2003/0232056 A1 (SEQ IDNO:215); US 2004/0044179 A1 (SEQ ID NO:16); US 2004/0044180 A1 (SEQ IDNO:16); US 2005/0238649 A1 (referring to human TAT226 as “PSCA Hlog”);WO 2003/025148 (SEQ ID NO:292); WO 2003/105758 (SEQ ID NO:14); and EP1347046 (SEQ ID NO:2640).

Full-length TAT226 undergoes processing in the cell to generate a matureform of the protein that is expressed on the cell surface. For example,a full length human TAT226 as shown in SEQ ID NO:75 contains a predictedsignal peptide sequence from amino acids 1-20 or 1-22, which ispredicted to be cleaved from the protein. The C-terminus from aminoacids 116-141 is predicted to be cleaved from the protein, and a GPImoiety attached to the protein at amino acid 115. A mature form of humanTAT226, from amino acids 21-115 or 23-115 of SEQ ID NO:75, is predictedto be anchored to the cell surface via the GPI moiety. Monkey and rodentTAT226 (see, e.g., SEQ ID NOs:76-78) are highly similar to human TAT226and thus are believed to be cleaved and modified at the equivalent aminoacid positions. See FIG. 1. The resulting mature forms of human, monkey,and rodent TAT226 from amino acid 21-115 or 23-115 (as shown in FIG. 1)are 100% identical.

Other features of human TAT226 include a predicted N-glycosylation siteat amino acid 45, which was confirmed empirically, and a predictedLy6/u-PAR domain from amino acids 94-107 of SEQ ID NO:75. Human TAT226has about 32% amino acid homology with prostate stem cell antigen(PSCA), a prostate cancer-specific tumor antigen that is expressed onthe cell surface via a GPI linkage. See Reiter et al. (1998) Proc.Nat'l. Acad. Sci. USA 95:1735-1740. PSCA is overexpressed in over 80% ofprostate cancers. Id. Like TAT226, it contains a predicted Ly6/u-PARdomain, which has been implicated in cell functions such as signaltransduction and cell adhesion. Id.

In one aspect, the invention provides antibodies that bind to TAT226. Insome embodiments, antibodies are provided that bind to a mature form ofTAT226. In one such embodiment, a mature form of TAT226 has an aminoacid sequence from amino acid 21-115 or 23-115 of SEQ ID NO:75. In someembodiments, an antibody to TAT226 binds to a mature form of TAT226expressed on the cell surface. In some embodiments, an antibody thatbinds to a mature form of TAT226 expressed on the cell surface inhibitsthe growth of a cell. In some embodiments, an anti-TAT226 antibody bindsto a mature form of TAT226 expressed on the cell surface and inhibitscell proliferation. In certain embodiments, an anti-TAT226 antibodybinds to a mature form of TAT226 expressed on the cell surface andinduces cell death. In some embodiments, an anti-TAT226 antibody bindsto a mature form of TAT226 expressed on the surface of cancer cells. Insome embodiments, an anti-TAT226 antibody binds to a mature form ofTAT226 that is overexpressed on the surface of cancer cells relative tonormal cells of the same tissue origin.

In one aspect, an anti-TAT226 antibody is a monoclonal antibody. In oneaspect, an anti-TAT226 antibody is an antibody fragment, e.g., a Fab,Fab′-SH, Fv, scFv, or (Fab′)₂ fragment. In one aspect, an anti-TAT226antibody is a chimeric, humanized, or human antibody. In one aspect, anyof the anti-TAT226 antibodies described herein are purified.

Exemplary monoclonal antibodies derived from a phage library areprovided herein as described in Example B. The antigen used forscreening the library was a polypeptide having the sequence of aminoacids 1-115 of SEQ ID NO:75, corresponding to a form of TAT226 lackingthe amino acids that are C-terminal of the putative GPI attachment site.The antibodies resulting from the library screen are designated YWO.32and YWO.49. YWO.49 was affinity matured to generate YWO.49.B7,YWO.49.C9, YWO.49.H2, and YWO.49.H6. Alignments of the sequences of theheavy and light chain variable domains of YWO.32, YWO.49, YWO.49.B7,YWO.49.C9, YWO.49.H2, and YWO.49.H6 are shown in FIGS. 11 and 12,respectively.

In one aspect, monoclonal antibodies that compete with YWO.32, YWO.49,YWO.49.B7, YWO.49.C9, YWO.49.H2, or YWO.49.H6 for binding to TAT226 areprovided. Monoclonal antibodies that bind to the same epitope as YWO.32,YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2, or YWO.49.H6 are also provided.

In one aspect of the invention, polynucleotides encoding anti-TAT226antibodies are provided. In certain embodiments, vectors comprisingpolynucleotides encoding anti-TAT226 antibodies are provided. In certainembodiments, host cells comprising such vectors are provided. In anotheraspect of the invention, compositions comprising anti-TAT226 antibodiesor polynucleotides encoding anti-TAT226 antibodies are provided. Incertain embodiments, a composition of the invention is a pharmaceuticalformulation for the treatment of a cell proliferative disorder, such asthose enumerated herein.

A detailed description of exemplary anti-TAT226 antibodies is asfollows:

1. Specific Embodiments of Anti-TAT226 Antibodies

In one aspect, the invention provides an antibody comprising at leastone, two, three, four, five, or six HVRs selected from (a) an HVR-H1comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:4; (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5;(c) an HVR-H3 comprising an amino acid sequence selected from SEQ IDNO:3 and 6-11; (d) an HVR-L1 comprising the amino acid sequence of SEQID NO:12; (e) an HVR-L2 comprising the amino acid sequence of SEQ IDNO:13; and (f) an HVR-L3 comprising an amino acid sequence selected fromSEQ ID NO:14-19.

In one aspect, the invention provides an anti-TAT226 antibody comprisingat least one, at least two, or all three VH HVR sequences selected from(a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:1 or SEQID NO:4; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:2or SEQ ID NO:5; and (c) an HVR-H3 comprising an amino acid sequenceselected from SEQ ID NO:3 and 6-11. In one aspect, the inventionprovides an anti-TAT226 antibody comprising an HVR-H1 comprising theamino acid sequence of SEQ ID NO:1 or SEQ ID NO:4. In one aspect, theinvention provides an anti-TAT226 antibody comprising an HVR-H2comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5. In oneaspect, the invention provides an anti-TAT226 antibody comprising anHVR-H3 comprising an amino acid sequence selected from SEQ ID NO:3 and6-11. In one aspect the invention provides an anti-TAT226 antibodycomprising an HVR-H3 comprising the amino acid sequence of SEQ ID NO:9or 10.

In one aspect, the invention provides an anti-TAT226 antibody comprisingan HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:3and 6-11 and an HVR-H1 comprising an amino acid sequence selected fromSEQ ID NO:1 and SEQ ID NO:4. In one aspect, the invention provides ananti-TAT226 antibody comprising an HVR-H3 comprising an amino acidsequence selected from SEQ ID NO:6-11 and an HVR-H1 comprising SEQ IDNO:4. In one aspect, the invention provides an anti-TAT226 antibodycomprising an HVR-H3 comprising SEQ ID NO:9 or 10 and an HVR-H1comprising SEQ ID NO:4. In one aspect, the invention provides ananti-TAT226 antibody comprising an HVR-H3 comprising SEQ ID NO:3 and anHVR-H1 comprising SEQ ID NO:1.

In one aspect, the invention provides an anti-TAT226 antibody comprisingan HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:3and 6-11 and an HVR-H2 comprising an amino acid sequence selected fromSEQ ID NO:2 and SEQ ID NO:5. In one aspect, the invention provides ananti-TAT226 antibody comprising an HVR-H3 comprising an amino acidsequence selected from SEQ ID NO:6-11 and an HVR-H2 comprising SEQ IDNO:5. In one aspect, the invention provides an anti-TAT226 antibodycomprising an HVR-H3 comprising SEQ ID NO:9 or 10 and an HVR-H2comprising SEQ ID NO:5. In one aspect, the invention provides ananti-TAT226 antibody comprising an HVR-H3 comprising SEQ ID NO:3 and anHVR-H2 comprising SEQ ID NO:2.

In one aspect, the invention provides an anti-TAT226 antibody comprisingan HVR-H1 comprising the amino acid sequence of SEQ ID NO:1 or SEQ IDNO:4; an HVR-H2 comprising the amino acid sequence of SEQ ID NO:2 or SEQID NO:5; and an HVR-H3 comprising an amino acid sequence selected fromSEQ ID NO:3 and 6-11. In one embodiment, the HVR-H1 comprises the aminoacid sequence of SEQ ID NO:4; the HVR-H2 comprises the amino acidsequence of SEQ ID NO:5; and the HVR-H3 comprises an amino acid sequenceselected from SEQ ID NO:6-11. In one embodiment, the HVR-H1 comprisesthe amino acid sequence of SEQ ID NO:4; the HVR-H2 comprises the aminoacid sequence of SEQ ID NO:5; and the HVR-H3 comprises SEQ ID NO:9 or10. In one embodiment, the HVR-H1 comprises the amino acid sequence ofSEQ ID NO:1; the HVR-H2 comprises the amino acid sequence of SEQ IDNO:2; and the HVR-H3 comprises the amino acid sequence of SEQ ID NO:3.

In one aspect, the invention provides an anti-TAT226 antibody comprisingat least one, at least two, or all three VL HVR sequences selected from(a) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:12; (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO:13; and (c) anHVR-H3 comprising an amino acid sequence selected from SEQ ID NO:14-19.In one aspect, the invention provides an anti-TAT226 antibody comprisingan HVR-L3 comprising an amino acid sequence selected from SEQ IDNO:14-19. In one aspect, the invention provides an anti-TAT226 antibodycomprising an HVR-L3 comprising the amino acid sequence of SEQ ID NO:17or 18.

In one aspect, the invention provides an anti-TAT226 antibody comprising(a) an HVR-H3 comprising an amino acid sequence selected from SEQ IDNO:3 and 6-11 and (b) an HVR-L3 comprising an amino acid sequenceselected from SEQ ID NO:14-19. In one aspect, the invention provides ananti-TAT226 antibody comprising (a) an HVR-H3 comprising the amino acidsequence of SEQ ID NO:3 and (b) an HVR-L3 comprising the amino acidsequence of SEQ ID NO:14. In some embodiments, the TAT226 antibodyfurther comprises (a) an HVR-H1 comprising SEQ ID NO:1 and an HVR-H2comprising SEQ ID NO:2.

In one aspect, the invention provides an anti-TAT226 antibody comprising(a) an HVR-H3 comprising an amino acid sequence selected from SEQ IDNO:6-11 and (b) an HVR-L3 comprising an amino acid sequence selectedfrom SEQ ID NO:14-19. In some embodiments, the HVR-H3 comprises theamino acid sequence of SEQ ID NO:9 and the HVR-L3 comprises the aminoacid sequence of SEQ ID NO:17. In some embodiments, the HVR-H3 comprisesthe amino acid sequence of SEQ ID NO:10 and the HVR-L3 comprises theamino acid sequence of SEQ ID NO:18. In some embodiments, the TAT226antibody further comprises an HVR-H1 comprising SEQ ID NO:4 and anHVR-H2 comprising SEQ ID NO:5.

In one aspect, the invention provides an anti-TAT226 antibody comprising(a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:1 or SEQID NO:4; (b) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:2or SEQ ID NO:5; (c) an HVR-H3 comprising an amino acid sequence selectedfrom SEQ ID NO:3 and 6-11; (d) an HVR-L1 comprising the amino acidsequence of SEQ ID NO:12; (e) an HVR-L2 comprising the amino acidsequence of SEQ ID NO:13; and (f) an HVR-L3 comprising an amino acidsequence selected from SEQ ID NO:14-19.

In one aspect, the invention provides an anti-TAT226 antibody comprising(a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO:2; (c) an HVR-H3comprising the amino acid sequence of SEQ ID NO:3; (d) an HVR-L1comprising the amino acid sequence of SEQ ID NO:12; (e) an HVR-L2comprising the amino acid sequence of SEQ ID NO:13; and (f) an HVR-L3comprising the amino acid sequence of SEQ ID NO:14.

In one aspect, the invention provides an anti-TAT226 antibody comprising(a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:4; (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO:5; (c) an HVR-H3comprising an amino acid sequence selected from SEQ ID NO:6-11; (d) anHVR-L1 comprising the amino acid sequence of SEQ ID NO:12; (e) an HVR-L2comprising the amino acid sequence of SEQ ID NO:13; and (f) an HVR-L3comprising an amino acid sequence selected from SEQ ID NO:14-19.

In one aspect, the invention provides an anti-TAT226 antibody comprising(a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:4; (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO:5; (c) an HVR-H3comprising the amino acid sequence of SEQ ID NO:9; (d) an HVR-L1comprising the amino acid sequence of SEQ ID NO:12; (e) an HVR-L2comprising the amino acid sequence of SEQ ID NO:13; and (f) an HVR-L3comprising the amino acid sequence of SEQ ID NO:17.

In one aspect, the invention provides an anti-TAT226 antibody comprising(a) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:4; (b) anHVR-H2 comprising the amino acid sequence of SEQ ID NO:5; (c) an HVR-H3comprising the amino acid sequence of SEQ ID NO:10; (d) an HVR-L1comprising the amino acid sequence of SEQ ID NO:12; (e) an HVR-L2comprising the amino acid sequence of SEQ ID NO:13; and (f) an HVR-L3comprising the amino acid sequence of SEQ ID NO:18.

In some embodiments, an anti-TAT226 antibody is affinity matured toobtain the desired target binding affinity. In some embodiments, any oneor more amino acids of an antibody are substituted at the following HVRpositions (Kabat numbered): H98, H99, H100, H100B, L90, L92, L93, L96,and L97. For example, in some embodiments, any one or more of thefollowing substitutions may be made in any combination:

-   -   in HVR-H3 (SEQ ID NO:6): V98I; S99T; R100L or I; G100bA, S, or P    -   in HVR-L3 (SEQ ID NO:14): Q90R, K, H, or N; Y92V; T93F, N, G, or        A; P96F; T97I or A        All possible combinations of the above substitutions are        encompassed by the consensus sequences of SEQ ID NO:11 (HVR-H3)        and SEQ ID NO:19 (HVR-L3).

An anti-TAT226 antibody may comprise any suitable framework variabledomain sequence, provided that the antibody retains the ability to bindTAT226. For example, in some embodiments, anti-TAT226 antibodies of theinvention comprise a human subgroup III heavy chain framework consensussequence. In one embodiment of these antibodies, the heavy chainframework consensus sequence comprises substitution(s) at position 71,73 and/or 78. In one embodiments of these antibodies, position 71 is A,position 73 is T, and/or position 78 is A. In one embodiment, theseantibodies comprise a heavy chain variable domain framework sequence ofhuMAb4D5-8, e.g., SEQ ID NO:50, 51, 59, 35 (FR1, 2, 3, 4, respectively).huMAb4D5-8 is commercially known as HERCEPTIN®, Genentech, Inc., SouthSan Francisco, Calif., USA; also referred to in U.S. Pat. Nos. 6,407,213& 5,821,337, and Lee et al., J. Mol. Biol. (2004), 340(5):1073-93. Inone such embodiment, these antibodies further comprise a human id lightchain framework consensus sequence. In one such embodiment, theseantibodies comprise a light chain variable domain framework sequence ofhuMAb4D5-8.

In one embodiment, an anti-TAT226 antibody comprises a heavy chainvariable domain comprising a framework sequence and hypervariableregions, wherein the framework sequence comprises the FR1-FR4 sequencesselected from those shown in FIGS. 5A and 5B; the HVR H1 comprises theamino acid sequence of SEQ ID NO:4; the HVR-H2 comprises the amino acidsequence of SEQ ID NO:5; and the HVR-H3 comprises an amino acid sequenceselected from SEQ ID NO:6-11. In one embodiment of these antibodies, theHVR-H3 comprises the amino acid sequence of SEQ ID NO:9 or 10. In oneembodiment, an anti-TAT226 antibody comprises a light chain variabledomain comprising a framework sequence and hypervariable regions,wherein the framework sequence comprises the FR1-FR4 sequences selectedfrom those shown in FIGS. 6A and 6B; the HVR-L1 comprises the amino acidsequence of SEQ ID NO:12; the HVR-L2 comprises the amino acid sequenceof SEQ ID NO:13; and the HVR-L3 comprises an amino acid sequenceselected from SEQ ID NO:14-19. In one embodiment of these antibodies,the HVR-L3 comprises the amino acid sequence of SEQ ID NO:17 or 18.

In one embodiment, an anti-TAT226 antibody comprises a heavy chainvariable domain comprising a framework sequence and hypervariableregions, wherein the framework sequence comprises the FR1-FR4 sequencesof SEQ ID NO:50, 51, 59, and 35, as shown in FIG. 7; the HVR H1comprises the amino acid sequence of SEQ ID NO:4; the HVR-H2 comprisesthe amino acid sequence of SEQ ID NO:5; and the HVR-H3 comprises anamino acid sequence selected from SEQ ID NO:6-11. In one embodiment ofthese antibodies, the HVR-H3 comprises the amino acid sequence of SEQ IDNO:9 or 10. In one embodiment, an anti-TAT226 antibody comprises a lightchain variable domain comprising a framework sequence and hypervariableregions, wherein the framework sequence comprises the FR1-FR4 sequencesof SEQ ID NO:60, 61, 62, and 63, as shown in FIGS. 6A and 6B; the HVR-L1comprises the amino acid sequence of SEQ ID NO:12; the HVR-L2 comprisesthe amino acid sequence of SEQ ID NO:13; and the HVR-L3 comprises anamino acid sequence selected from SEQ ID NO:14-19. In one embodiment ofthese antibodies, the HVR-L3 comprises the amino acid sequence of SEQ IDNO:17 or 18.

In one embodiment, an anti-TAT226 antibody comprises a heavy chainvariable domain comprising a framework sequence and hypervariableregions, wherein the framework sequence comprises the FR1-FR4 sequencesof SEQ ID NO:50, 51, 53, and 35, as shown in FIG. 8; the HVR H1comprises the amino acid sequence of SEQ ID NO:4; the HVR-H2 comprisesthe amino acid sequence of SEQ ID NO:5; and the HVR-H3 comprises anamino acid sequence selected from SEQ ID NO:6-11. In one embodiment ofthese antibodies, the HVR-H3 comprises the amino acid sequence of SEQ IDNO:9 or 10. In one embodiment, an anti-TAT226 antibody comprises a lightchain variable domain comprising a framework sequence and hypervariableregions, wherein the framework sequence comprises the FR1-FR4 sequencesof SEQ ID NO:60, 61, 62, and 74, as shown in FIG. 8; the HVR-L1comprises the amino acid sequence of SEQ ID NO:12; the HVR-L2 comprisesthe amino acid sequence of SEQ ID NO:13; and the HVR-L3 comprises anamino acid sequence selected from SEQ ID NO:14-19. In one embodiment ofthese antibodies, the HVR-L3 comprises the amino acid sequence of SEQ IDNO:17 or 18.

In some embodiments, the invention provides an anti-TAT226 antibodycomprising a heavy chain variable domain comprising an amino acidsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity to an amino acid sequence selected from SEQ IDNO:20-25. In some embodiments, an amino acid sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identitycontains substitutions, insertions, or deletions relative to thereference sequence, but an antibody comprising that amino acid sequenceretains the ability to bind to TAT226. In some embodiments, a total of 1to 10 amino acids have been substituted, inserted, or deleted in asequence selected from SEQ ID NO:20-25. In some embodiments, thesubstitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). In some embodiments, an anti-TAT226 antibodycomprises a heavy chain variable domain comprising an amino acidsequence selected from SEQ ID NO:20-25.

In some embodiments, the invention provides an anti-TAT226 antibodycomprising a light chain variable domain of humanized 4D5 antibody(huMAb4D5-8) (HERCEPTIN®, Genentech, Inc., South San Francisco, Calif.,USA) (also referred to in U.S. Pat. No. 6,407,213 and Lee et al., J.Mol. Biol. (2004), 340(5):1073-93) as depicted in SEQ ID NO:31 below.

(SEQ ID NO: 31)1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr IleThr Cys Arg Ala Ser Gln Asp Val 

 Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly LysAla Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe SerGly Ser 

 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe AlaThr Tyr Tyr Cys Gln Gln 

 Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 108(HVR residues are underlined)

In some embodiments, the huMAb4D5-8 light chain variable domain sequenceis modified at one or more of positions 30, 66 and 91 (Asn, Arg and Hisas indicated in bold/italics above, respectively). In one embodiment,the modified huMAb4D5-8 sequence comprises Ser in position 30, Gly inposition 66 and/or Ser in position 91. Accordingly, in one embodiment,an antibody of the invention comprises a light chain variable domaincomprising the sequence depicted in SEQ ID NO:26 below:

(SEQ ID NO: 26)1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr IleThr Cys Arg Ala Ser Gln Asp Val 

 Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly LysAla Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe SerGly Ser 

 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe AlaThr Tyr Tyr Cys Gln Gln 

 Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 108(HVR residues are underlined)Substituted residues with respect to huMAb4D5-8 are indicated inbold/italics above.

In one aspect, the invention provides an anti-TAT226 antibody comprisinga light chain variable domain comprising an amino acid sequence havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to an amino acid sequence selected from SEQ ID NO:26-31. Insome embodiments, an amino acid sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity containssubstitutions, additions, or deletions relative to the referencesequence, but an antibody comprising that amino acid sequence retainsthe ability to bind to TAT226. In some embodiments, a total of 1 to 10amino acids have been substituted, inserted, or deleted in a sequenceselected from SEQ ID NO:26-31. In some embodiments, the substitutions,insertions, or deletions occur in regions outside the HVRs (i.e., in theFRs). In some embodiments, an anti-TAT226 antibody comprises a lightchain variable domain comprising an amino acid sequence selected fromSEQ ID NO:26-31.

In one aspect, the invention provides an anti-TAT226 antibody comprising(a) a heavy chain variable domain comprising an amino acid sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to an amino acid sequence selected from SEQ IDNO:20-25; and (b) a light chain variable domain comprising an amino acidsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity to an amino acid sequence selected from SEQ IDNO:26-31. In some embodiments, an amino acid sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identitycontains substitutions, additions, or deletions relative to thereference sequence, but an antibody comprising that amino acid sequenceretains the ability to bind to TAT226. In some embodiments, a total of 1to 10 amino acids have been substituted, inserted, or deleted in thereference sequence. In some embodiments, the substitutions, insertions,or deletions occur in regions outside the HVRs (i.e., in the FRs). Insome embodiments, an anti-TAT226 antibody comprises a heavy chainvariable domain comprising an amino acid sequence selected from SEQ IDNO:20-25 and a light chain variable domain comprising an amino acidsequence selected from SEQ ID NO:26-31.

In some embodiments, an anti-TAT226 antibody comprises a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO:20 and alight chain variable region comprising the amino acid sequence of SEQ IDNO:26. In some embodiments, an anti-TAT226 antibody comprises a heavychain variable region comprising the amino acid sequence of SEQ ID NO:21and a light chain variable region comprising the amino acid sequence ofSEQ ID NO:26. In some embodiments, an anti-TAT226 antibody comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO:22 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:27. In some embodiments, an anti-TAT226 antibodycomprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO:23 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO:28. In some embodiments, ananti-TAT226 antibody comprises a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO:24 and a light chain variableregion comprising the amino acid sequence of SEQ ID NO:29. In someembodiments, an anti-TAT226 antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:25 and a lightchain variable region comprising the amino acid sequence of SEQ IDNO:30.

In one aspect, the invention provides an anti-TAT226 antibody comprising(a) one, two, or three VH HVRs selected from those shown in FIGS. 2 and4 and/or (b) one, two, or three VL HVRs selected from those shown inFIGS. 3 and 4. In one aspect, the invention provides an anti-TAT226antibody comprising a heavy chain variable domain selected from thoseshown in FIGS. 9 and 11 and a light chain variable domain selected fromthose shown in FIGS. 10 and 12.

2. Antibody Fragments

The present invention encompasses antibody fragments. Antibody fragmentsmay be generated by traditional means, such as enzymatic digestion, orby recombinant techniques. In certain circumstances there are advantagesof using antibody fragments, rather than whole antibodies. The smallersize of the fragments allows for rapid clearance, and may lead toimproved access to solid tumors. For a review of certain antibodyfragments, see Hudson et al. (2003) Nat. Med. 9:129-134.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 (1992)). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)₂ fragment with increased in vivohalf-life comprising salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In certain embodiments, an antibody is a single chain Fvfragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and5,587,458. Fv and scFv are the only species with intact combining sitesthat are devoid of constant regions; thus, they may be suitable forreduced nonspecific binding during in vivo use. scFv fusion proteins maybe constructed to yield fusion of an effector protein at either theamino or the carboxy terminus of an scFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870, for example.Such linear antibodies may be monospecific or bispecific.

3. Humanized Antibodies

The invention encompasses humanized antibodies. Various methods forhumanizing non-human antibodies are known in the art. For example, ahumanized antibody can have one or more amino acid residues introducedinto it from a source which is non-human. These non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.(1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327;Verhoeyen et al. (1988) Science 239:1534-1536), by substitutinghypervariable region sequences for the corresponding sequences of ahuman antibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567) wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some hypervariable region residuesand possibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies can be important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework for the humanized antibody (Sims et al. (1993) J.Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901. Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285;Presta et al. (1993) J. Immunol., 151:2623.

It is further generally desirable that antibodies be humanized withretention of high affinity for the antigen and other favorablebiological properties. To achieve this goal, according to one method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

4. Human Antibodies

Human anti-TAT226 antibodies of the invention can be constructed bycombining Fv clone variable domain sequence(s) selected fromhuman-derived phage display libraries with known human constant domainsequences(s) as described above. Alternatively, human monoclonalanti-TAT226 antibodies of the invention can be made by the hybridomamethod. Human myeloma and mouse-human heteromyeloma cell lines for theproduction of human monoclonal antibodies have been described, forexample, by Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol.,147: 86 (1991).

It is now possible to produce transgenic animals (e.g. mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (JH) gene in chimeric and germ-linemutant mice results in complete inhibition of endogenous antibodyproduction. Transfer of the human germ-line immunoglobulin gene array insuch germ-line mutant mice will result in the production of humanantibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol., 7: 33 (1993).

Gene shuffling can also be used to derive human antibodies fromnon-human, e.g. rodent, antibodies, where the human antibody has similaraffinities and specificities to the starting non-human antibody.According to this method, which is also called “epitope imprinting”,either the heavy or light chain variable region of a non-human antibodyfragment obtained by phage display techniques as described herein isreplaced with a repertoire of human V domain genes, creating apopulation of non-human chain/human chain scFv or Fab chimeras.Selection with antigen results in isolation of a non-human chain/humanchain chimeric scFv or Fab wherein the human chain restores the antigenbinding site destroyed upon removal of the corresponding non-human chainin the primary phage display clone, i.e. the epitope governs (imprints)the choice of the human chain partner. When the process is repeated inorder to replace the remaining non-human chain, a human antibody isobtained (see PCT WO 93/06213 published Apr. 1, 1993). Unliketraditional humanization of non-human antibodies by CDR grafting, thistechnique provides completely human antibodies, which have no FR or CDRresidues of non-human origin.

5. Bispecific Antibodies

Bispecific antibodies are monoclonal antibodies that have bindingspecificities for at least two different antigens. In certainembodiments, bispecific antibodies are human or humanized antibodies. Incertain embodiments, one of the binding specificities is for TAT226 andthe other is for any other antigen. In certain embodiments, bispecificantibodies may bind to two different epitopes of TAT226. Bispecificantibodies may also be used to localize cytotoxic agents to cells whichexpress TAT226. These antibodies possess a TAT226-binding arm and an armwhich binds a cytotoxic agent, such as, e.g., saporin,anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate orradioactive isotope hapten. Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab′)₂ bispecificantibodies).

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305: 537 (1983)). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. The purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829 published May 13, 1993, and inTraunecker et al., EMBO J., 10: 3655 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion, forexample, is with an immunoglobulin heavy chain constant domain,comprising at least part of the hinge, CH2, and CH3 regions. In certainembodiments, the first heavy-chain constant region (CH1), containing thesite necessary for light chain binding, is present in at least one ofthe fusions. DNAs encoding the immunoglobulin heavy chain fusions and,if desired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In one embodiment of this approach, the bispecific antibodies arecomposed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach, the interface between a pair of antibodymolecules can be engineered to maximize the percentage of heterodimerswhich are recovered from recombinant cell culture. The interfacecomprises at least a part of the C_(H)3 domain of an antibody constantdomain. In this method, one or more small amino acid side chains fromthe interface of the first antibody molecule are replaced with largerside chains (e.g. tyrosine or tryptophan). Compensatory “cavities” ofidentical or similar size to the large side chain(s) are created on theinterface of the second antibody molecule by replacing large amino acidside chains with smaller ones (e.g. alanine or threonine). This providesa mechanism for increasing the yield of the heterodimer over otherunwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/00373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking method. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the HER2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (VH) connected to a light-chain variabledomain (VL) by a linker which is too short to allow pairing between thetwo domains on the same chain. Accordingly, the VH and VL domains of onefragment are forced to pair with the complementary VL and VH domains ofanother fragment, thereby forming two antigen-binding sites. Anotherstrategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See Gruber et al.,J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

6. Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present invention can bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen binding sites (e.g. tetravalent antibodies), whichcan be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or more antigenbinding sites. In certain embodiments, the dimerization domain comprises(or consists of) an Fc region or a hinge region. In this scenario, theantibody will comprise an Fc region and three or more antigen bindingsites amino-terminal to the Fc region. In certain embodiments, amultivalent antibody comprises (or consists of) three to about eightantigen binding sites. In one such embodiment, a multivalent antibodycomprises (or consists of) four antigen binding sites. The multivalentantibody comprises at least one polypeptide chain (for example, twopolypeptide chains), wherein the polypeptide chain(s) comprise two ormore variable domains. For instance, the polypeptide chain(s) maycomprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain,VD2 is a second variable domain, Fc is one polypeptide chain of an Fcregion, X1 and X2 represent an amino acid or polypeptide, and n is 0or 1. For instance, the polypeptide chain(s) may comprise:VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fcregion chain. The multivalent antibody herein may further comprise atleast two (for example, four) light chain variable domain polypeptides.The multivalent antibody herein may, for instance, comprise from abouttwo to about eight light chain variable domain polypeptides. The lightchain variable domain polypeptides contemplated here comprise a lightchain variable domain and, optionally, further comprise a CL domain.

7. Single-Domain Antibodies

In some embodiments, an antibody of the invention is a single-domainantibody. A single-domain antibody is a single polypeptide chaincomprising all or a portion of the heavy chain variable domain or all ora portion of the light chain variable domain of an antibody. In certainembodiments, a single-domain antibody is a human single-domain antibody(Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).In one embodiment, a single-domain antibody consists of all or a portionof the heavy chain variable domain of an antibody.

8. Antibody Variants

In some embodiments, amino acid sequence modification(s) of theantibodies described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody Amino acid sequence variants of the antibodymay be prepared by introducing appropriate changes into the nucleotidesequence encoding the antibody, or by peptide synthesis. Suchmodifications include, for example, deletions from, and/or insertionsinto and/or substitutions of, residues within the amino acid sequencesof the antibody. Any combination of deletion, insertion, andsubstitution can be made to arrive at the final construct, provided thatthe final construct possesses the desired characteristics. The aminoacid alterations may be introduced in the subject antibody amino acidsequence at the time that sequence is made.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells (1989)Science, 244:1081-1085. Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (e.g.,alanine or polyalanine) to affect the interaction of the amino acidswith antigen. Those amino acid locations demonstrating functionalsensitivity to the substitutions then are refined by introducing furtheror other variants at, or for, the sites of substitution. Thus, while thesite for introducing an amino acid sequence variation is predetermined,the nature of the mutation per se need not be predetermined. Forexample, to analyze the performance of a mutation at a given site, alascanning or random mutagenesis is conducted at the target codon orregion and the expressed immunoglobulins are screened for the desiredactivity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

In certain embodiments, an antibody of the invention is altered toincrease or decrease the extent to which the antibody is glycosylated.Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of a carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition or deletion of glycosylation sites to the antibody isconveniently accomplished by altering the amino acid sequence such thatone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites) is created or removed. The alteration may also bemade by the addition, deletion, or substitution of one or more serine orthreonine residues to the sequence of the original antibody (forO-linked glycosylation sites).

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. For example, antibodies with a maturecarbohydrate structure that lacks fucose attached to an Fc region of theantibody are described in US Pat Appl No US 2003/0157108 (Presta, L.).See also US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies with abisecting N-acetylglucosamine (GlcNAc) in the carbohydrate attached toan Fc region of the antibody are referenced in WO 2003/011878,Jean-Mairet et al. and U.S. Pat. No. 6,602,684, Umana et al. Antibodieswith at least one galactose residue in the oligosaccharide attached toan Fc region of the antibody are reported in WO 1997/30087, Patel et al.See, also, WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.)concerning antibodies with altered carbohydrate attached to the Fcregion thereof. See also US 2005/0123546 (Umana et al.) onantigen-binding molecules with modified glycosylation.

In certain embodiments, a glycosylation variant comprises an Fc region,wherein a carbohydrate structure attached to the Fc region lacks fucose.Such variants have improved ADCC function. Optionally, the Fc regionfurther comprises one or more amino acid substitutions therein whichfurther improve ADCC, for example, substitutions at positions 298, 333,and/or 334 of the Fc region (Eu numbering of residues). Examples ofpublications related to “defucosylated” or “fucose-deficient” antibodiesinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; Okazaki et al. J. Mol. Biol.336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614(2004). Examples of cell lines producing defucosylated antibodiesinclude Lec13 CHO cells deficient in protein fucosylation (Ripka et al.Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,especially at Example 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells(Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)).

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. Sites of interest for substitutionalmutagenesis include the hypervariable regions, but FR alterations arealso contemplated. Conservative substitutions are shown in Table 1 underthe heading of “preferred substitutions.” If such substitutions resultin a desirable change in biological activity, then more substantialchanges, denominated “exemplary substitutions” in Table 1, or as furtherdescribed below in reference to amino acid classes, may be introducedand the products screened.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp; Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Leu Norleucine Leu (L) Norleucine;Ile; Val; Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain Amino acids may begrouped according to similarities in the properties of their side chains(in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, WorthPublishers, New York (1975)):

(1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp(W), Met (M)

(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn(N), Gln (Q)

(3) acidic: Asp (D), Glu (E)

(4) basic: Lys (K), Arg (R), His (H)

Alternatively, naturally occurring residues may be divided into groupsbased on common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, into theremaining (non-conserved) sites.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther development will have modified (e.g., improved) biologicalproperties relative to the parent antibody from which they aregenerated. A convenient way for generating such substitutional variantsinvolves affinity maturation using phage display. Briefly, severalhypervariable region sites (e.g. 6-7 sites) are mutated to generate allpossible amino acid substitutions at each site. The antibodies thusgenerated are displayed from filamentous phage particles as fusions toat least part of a phage coat protein (e.g., the gene III product ofM13) packaged within each particle. The phage-displayed variants arethen screened for their biological activity (e.g. binding affinity). Inorder to identify candidate hypervariable region sites for modification,scanning mutagenesis (e.g., alanine scanning) can be performed toidentify hypervariable region residues contributing significantly toantigen binding. Alternatively, or additionally, it may be beneficial toanalyze a crystal structure of the antigen-antibody complex to identifycontact points between the antibody and antigen. Such contact residuesand neighboring residues are candidates for substitution according totechniques known in the art, including those elaborated herein. Oncesuch variants are generated, the panel of variants is subjected toscreening using techniques known in the art, including those describedherein, and antibodies with superior properties in one or more relevantassays may be selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications inan Fc region of antibodies of the invention, thereby generating an Fcregion variant. The Fc region variant may comprise a human Fc regionsequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprisingan amino acid modification (e.g. a substitution) at one or more aminoacid positions including that of a hinge cysteine.

In accordance with this description and the teachings of the art, it iscontemplated that in some embodiments, an antibody of the invention maycomprise one or more alterations as compared to the wild typecounterpart antibody, e.g. in the Fc region. These antibodies wouldnonetheless retain substantially the same characteristics required fortherapeutic utility as compared to their wild type counterpart. Forexample, it is thought that certain alterations can be made in the Fcregion that would result in altered (i.e., either improved ordiminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC),e.g., as described in WO99/51642. See also Duncan & Winter Nature322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; andWO94/29351 concerning other examples of Fc region variants. WO00/42072(Presta) and WO 2004/056312 (Lowman) describe antibody variants withimproved or diminished binding to FcRs. The content of these patentpublications are specifically incorporated herein by reference. See,also, Shields et al. J. Biol. Chem. 9(2): 6591-6604 (2001). Antibodieswith increased half lives and improved binding to the neonatal Fcreceptor (FcRn), which is responsible for the transfer of maternal IgGsto the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al.,J. Immunol. 24:249 (1994)), are described in US2005/0014934A1 (Hinton etal.). These antibodies comprise an Fc region with one or moresubstitutions therein which improve binding of the Fc region to FcRn.Polypeptide variants with altered Fc region amino acid sequences andincreased or decreased C1q binding capability are described in U.S. Pat.No. 6,194,551B1, WO99/51642. The contents of those patent publicationsare specifically incorporated herein by reference. See, also, Idusogieet al. J. Immunol. 164: 4178-4184 (2000).

In one aspect, the invention provides antibodies comprisingmodifications in the interface of Fc polypeptides comprising the Fcregion, wherein the modifications facilitate and/or promoteheterodimerization. These modifications comprise introduction of aprotuberance into a first Fc polypeptide and a cavity into a second Fcpolypeptide, wherein the protuberance is positionable in the cavity soas to promote complexing of the first and second Fc polypeptides.Methods of generating antibodies with these modifications are known inthe art, e.g., as described in U.S. Pat. No. 5,731,168.

9. Antibody Derivatives

The antibodies of the present invention can be further modified tocontain additional nonproteinaceous moieties that are known in the artand readily available. Preferably, the moieties suitable forderivatization of the antibody are water soluble polymers. Non-limitingexamples of water soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymer are attached, they can be the same ordifferent molecules. In general, the number and/or type of polymers usedfor derivatization can be determined based on considerations including,but not limited to, the particular properties or functions of theantibody to be improved, whether the antibody derivative will be used ina therapy under defined conditions, etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. 102: 11600-11605 (2005)).The radiation may be of any wavelength, and includes, but is not limitedto, wavelengths that do not harm ordinary cells, but which heat thenonproteinaceous moiety to a temperature at which cells proximal to theantibody-nonproteinaceous moiety are killed.

B. Certain Methods of Making Antibodies

1. Certain Hybridoma-Based Methods

The anti-TAT226 monoclonal antibodies of the invention can be made usingthe hybridoma method first described by Kohler et al., Nature, 256:495(1975), or may be made by recombinant DNA methods (U.S. Pat. No.4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theprotein used for immunization. Antibodies to TAT226 generally are raisedin animals by multiple subcutaneous (sc) or intraperitoneal (ip)injections of TAT226 and an adjuvant. TAT226 may be prepared usingmethods well-known in the art, some of which are further describedherein. For example, TAT226 may be produced recombinantly. In oneembodiment, animals are immunized with a derivative of TAT226 thatcontains an extracellular portion of TAT226 fused to the Fc portion ofan immunoglobulin heavy chain. In one embodiment, animals are immunizedwith an TAT226-IgG1 fusion protein. In one embodiment, animals areimmunized with immunogenic derivatives of TAT226 in a solution withmonophosphoryl lipid A (MPL)/trehalose dicrynomycolate (TDM) (RibiImmunochem. Research, Inc., Hamilton, Mont.), and the solution isinjected intradermally at multiple sites. Two weeks later the animalsare boosted. Seven to fourteen days later the animals are bled, and theserum is assayed for anti-TAT226 titer. Animals are boosted until titerplateaus.

Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium, e.g., a medium that contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

In certain embodiments, myeloma cells are those that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Exemplary myeloma cells include, but are not limited to, murinemyeloma lines, such as those derived from MOPC-21 and MPC-11 mousetumors available from the Salk Institute Cell Distribution Center, SanDiego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from theAmerican Type Culture Collection, Rockville, Md. USA. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001(1984); Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies that bind to TAT226. Preferably, thebinding specificity of monoclonal antibodies produced by hybridoma cellsis determined by immunoprecipitation or by an in vitro binding assay,such as radioimmunoassay (RIA) or enzyme-linked immunoadsorbent assay(ELISA). The binding affinity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis of Munson et al., Anal.Biochem., 107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.Monoclonal antibodies secreted by the subclones are suitably separatedfrom the culture medium, ascites fluid, or serum by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

2. Certain Library Screening Methods

Anti-TAT226 antibodies of the invention can be made by usingcombinatorial libraries to screen for antibodies with the desiredactivity or activities. For example, a variety of methods are known inthe art for generating phage display libraries and screening suchlibraries for antibodies possessing the desired binding characteristics.Such methods are described generally in Hoogenboom et al. (2001) inMethods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press,Totowa, N.J.), and in certain embodiments, in Lee et al. (2004) J. Mol.Biol. 340:1073-1093.

In principle, synthetic antibody clones are selected by screening phagelibraries containing phage that display various fragments of antibodyvariable region (Fv) fused to phage coat protein. Such phage librariesare panned by affinity chromatography against the desired antigen.Clones expressing Fv fragments capable of binding to the desired antigenare adsorbed to the antigen and thus separated from the non-bindingclones in the library. The binding clones are then eluted from theantigen, and can be further enriched by additional cycles of antigenadsorption/elution. Any of the anti-TAT226 antibodies of the inventioncan be obtained by designing a suitable antigen screening procedure toselect for the phage clone of interest followed by construction of afull length anti-TAT226 antibody clone using the Fv sequences from thephage clone of interest and suitable constant region (Fc) sequencesdescribed in Kabat et al., Sequences of Proteins of ImmunologicalInterest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991),vols. 1-3.

In certain embodiments, the antigen-binding domain of an antibody isformed from two variable (V) regions of about 110 amino acids, one eachfrom the light (VL) and heavy (VH) chains, that both present threehypervariable loops (HVRs) or complementarity-determining regions(CDRs). Variable domains can be displayed functionally on phage, eitheras single-chain Fv (scFv) fragments, in which VH and VL are covalentlylinked through a short, flexible peptide, or as Fab fragments, in whichthey are each fused to a constant domain and interact non-covalently, asdescribed in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Asused herein, scFv encoding phage clones and Fab encoding phage clonesare collectively referred to as “Fv phage clones” or “Fv clones.”

Repertoires of VH and VL genes can be separately cloned by polymerasechain reaction (PCR) and recombined randomly in phage libraries, whichcan then be searched for antigen-binding clones as described in Winteret al., Ann. Rev. Immunol., 12: 433-455 (1994). Libraries from immunizedsources provide high-affinity antibodies to the immunogen without therequirement of constructing hybridomas. Alternatively, the naiverepertoire can be cloned to provide a single source of human antibodiesto a wide range of non-self and also self antigens without anyimmunization as described by Griffiths et al., EMBO J, 12: 725-734(1993). Finally, naive libraries can also be made synthetically bycloning the unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

In certain embodiments, filamentous phage is used to display antibodyfragments by fusion to the minor coat protein pIII. The antibodyfragments can be displayed as single chain Fv fragments, in which VH andVL domains are connected on the same polypeptide chain by a flexiblepolypeptide spacer, e.g. as described by Marks et al., J. Mol. Biol.,222: 581-597 (1991), or as Fab fragments, in which one chain is fused topIII and the other is secreted into the bacterial host cell periplasmwhere assembly of a Fab-coat protein structure which becomes displayedon the phage surface by displacing some of the wild type coat proteins,e.g. as described in Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137(1991).

In general, nucleic acids encoding antibody gene fragments are obtainedfrom immune cells harvested from humans or animals. If a library biasedin favor of anti-TAT226 clones is desired, the subject is immunized withTAT226 to generate an antibody response, and spleen cells and/orcirculating B cells other peripheral blood lymphocytes (PBLs) arerecovered for library construction. In a preferred embodiment, a humanantibody gene fragment library biased in favor of anti-TAT226 clones isobtained by generating an anti-TAT226 antibody response in transgenicmice carrying a functional human immunoglobulin gene array (and lackinga functional endogenous antibody production system) such that TAT226immunization gives rise to B cells producing human antibodies againstTAT226. The generation of human antibody-producing transgenic mice isdescribed below.

Additional enrichment for anti-TAT226 reactive cell populations can beobtained by using a suitable screening procedure to isolate B cellsexpressing TAT226-specific membrane bound antibody, e.g., by cellseparation using TAT226 affinity chromatography or adsorption of cellsto fluorochrome-labeled TAT226 followed by flow-activated cell sorting(FACS).

Alternatively, the use of spleen cells and/or B cells or other PBLs froman unimmunized donor provides a better representation of the possibleantibody repertoire, and also permits the construction of an antibodylibrary using any animal (human or non-human) species in which TAT226 isnot antigenic. For libraries incorporating in vitro antibody geneconstruction, stem cells are harvested from the subject to providenucleic acids encoding unrearranged antibody gene segments. The immunecells of interest can be obtained from a variety of animal species, suchas human, mouse, rat, lagomorpha, luprine, canine, feline, porcine,bovine, equine, and avian species, etc.

Nucleic acid encoding antibody variable gene segments (including VH andVL segments) are recovered from the cells of interest and amplified. Inthe case of rearranged VH and VL gene libraries, the desired DNA can beobtained by isolating genomic DNA or mRNA from lymphocytes followed bypolymerase chain reaction (PCR) with primers matching the 5′ and 3′ endsof rearranged VH and VL genes as described in Orlandi et al., Proc.Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby making diverse Vgene repertoires for expression. The V genes can be amplified from cDNAand genomic DNA, with back primers at the 5′ end of the exon encodingthe mature V-domain and forward primers based within the J-segment asdescribed in Orlandi et al. (1989) and in Ward et al., Nature, 341:544-546 (1989). However, for amplifying from cDNA, back primers can alsobe based in the leader exon as described in Jones et al., Biotechnol.,9: 88-89 (1991), and forward primers within the constant region asdescribed in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-5732(1989). To maximize complementarity, degeneracy can be incorporated inthe primers as described in Orlandi et al. (1989) or Sastry et al.(1989). In certain embodiments, library diversity is maximized by usingPCR primers targeted to each V-gene family in order to amplify allavailable VH and VL arrangements present in the immune cell nucleic acidsample, e.g. as described in the method of Marks et al., J. Mol. Biol.,222: 581-597 (1991) or as described in the method of Orum et al.,Nucleic Acids Res., 21: 4491-4498 (1993). For cloning of the amplifiedDNA into expression vectors, rare restriction sites can be introducedwithin the PCR primer as a tag at one end as described in Orlandi et al.(1989), or by further PCR amplification with a tagged primer asdescribed in Clackson et al., Nature, 352: 624-628 (1991).

Repertoires of synthetically rearranged V genes can be derived in vitrofrom V gene segments. Most of the human VH-gene segments have beencloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227:776-798 (1992)), and mapped (reported in Matsuda et al., Nature Genet.,3: 88-94 (1993); these cloned segments (including all the majorconformations of the H1 and H2 loop) can be used to generate diverse VHgene repertoires with PCR primers encoding H3 loops of diverse sequenceand length as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). VH repertoires can also be made with all the sequencediversity focused in a long H3 loop of a single length as described inBarbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992). HumanVκ and Vλ segments have been cloned and sequenced (reported in Williamsand Winter, Eur. I Immunol., 23: 1456-1461 (1993)) and can be used tomake synthetic light chain repertoires. Synthetic V gene repertoires,based on a range of VH and VL folds, and L3 and H3 lengths, will encodeantibodies of considerable structural diversity. Following amplificationof V-gene encoding DNAs, germline V-gene segments can be rearranged invitro according to the methods of Hoogenboom and Winter, J. Mol. Biol.,227: 381-388 (1992).

Repertoires of antibody fragments can be constructed by combining VH andVL gene repertoires together in several ways. Each repertoire can becreated in different vectors, and the vectors recombined in vitro, e.g.,as described in Hogrefe et al., Gene, 128: 119-126 (1993), or in vivo bycombinatorial infection, e.g., the loxP system described in Waterhouseet al., Nucl. Acids Res., 21: 2265-2266 (1993). The in vivorecombination approach exploits the two-chain nature of Fab fragments toovercome the limit on library size imposed by E. coli transformationefficiency. Naive VH and VL repertoires are cloned separately, one intoa phagemid and the other into a phage vector. The two libraries are thencombined by phage infection of phagemid-containing bacteria so that eachcell contains a different combination and the library size is limitedonly by the number of cells present (about 10¹² clones). Both vectorscontain in vivo recombination signals so that the VH and VL genes arerecombined onto a single replicon and are co-packaged into phagevirions. These huge libraries provide large numbers of diverseantibodies of good affinity (K_(d) ⁻¹ of about 10⁻⁸M).

Alternatively, the repertoires may be cloned sequentially into the samevector, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci. USA,88: 7978-7982 (1991), or assembled together by PCR and then cloned, e.g.as described in Clackson et al., Nature, 352: 624-628 (1991). PCRassembly can also be used to join VH and VL DNAs with DNA encoding aflexible peptide spacer to form single chain Fv (scFv) repertoires. Inyet another technique, “in cell PCR assembly” is used to combine VH andVL genes within lymphocytes by PCR and then clone repertoires of linkedgenes as described in Embleton et al., Nucl. Acids Res., 20: 3831-3837(1992).

The antibodies produced by naive libraries (either natural or synthetic)can be of moderate affinity (K_(d) ⁻¹ of about 10⁶ to 10⁷ M⁻¹), butaffinity maturation can also be mimicked in vitro by constructing andreselecting from secondary libraries as described in Winter et al.(1994), supra. For example, mutation can be introduced at random invitro by using error-prone polymerase (reported in Leung et al.,Technique, 1: 11-15 (1989)) in the method of Hawkins et al., J. Mol.Biol., 226: 889-896 (1992) or in the method of Gram et al., Proc. Natl.Acad. Sci. USA, 89: 3576-3580 (1992). Additionally, affinity maturationcan be performed by randomly mutating one or more CDRs, e.g. using PCRwith primers carrying random sequence spanning the CDR of interest, inselected individual Fv clones and screening for higher affinity clones.WO 9607754 (published 14 Mar. 1996) described a method for inducingmutagenesis in a complementarity determining region of an immunoglobulinlight chain to create a library of light chain genes. Another effectiveapproach is to recombine the VH or VL domains selected by phage displaywith repertoires of naturally occurring V domain variants obtained fromunimmunized donors and screen for higher affinity in several rounds ofchain reshuffling as described in Marks et al., Biotechnol., 10: 779-783(1992). This technique allows the production of antibodies and antibodyfragments with affinities of about 10⁻⁹ M or less.

Screening of the libraries can be accomplished by various techniquesknown in the art. For example, TAT226 can be used to coat the wells ofadsorption plates, expressed on host cells affixed to adsorption platesor used in cell sorting, or conjugated to biotin for capture withstreptavidin-coated beads, or used in any other method for panning phagedisplay libraries.

The phage library samples are contacted with immobilized TAT226 underconditions suitable for binding at least a portion of the phageparticles with the adsorbent. Normally, the conditions, including pH,ionic strength, temperature and the like are selected to mimicphysiological conditions. The phages bound to the solid phase are washedand then eluted by acid, e.g. as described in Barbas et al., Proc. Natl.Acad. Sci USA, 88: 7978-7982 (1991), or by alkali, e.g. as described inMarks et al., J. Mol. Biol., 222: 581-597 (1991), or by TAT226 antigencompetition, e.g. in a procedure similar to the antigen competitionmethod of Clackson et al., Nature, 352: 624-628 (1991). Phages can beenriched 20-1,000-fold in a single round of selection. Moreover, theenriched phages can be grown in bacterial culture and subjected tofurther rounds of selection.

The efficiency of selection depends on many factors, including thekinetics of dissociation during washing, and whether multiple antibodyfragments on a single phage can simultaneously engage with antigen.Antibodies with fast dissociation kinetics (and weak binding affinities)can be retained by use of short washes, multivalent phage display andhigh coating density of antigen in solid phase. The high density notonly stabilizes the phage through multivalent interactions, but favorsrebinding of phage that has dissociated. The selection of antibodieswith slow dissociation kinetics (and good binding affinities) can bepromoted by use of long washes and monovalent phage display as describedin Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and alow coating density of antigen as described in Marks et al.,Biotechnol., 10: 779-783 (1992).

It is possible to select between phage antibodies of differentaffinities, even with affinities that differ slightly, for TAT226.However, random mutation of a selected antibody (e.g. as performed insome affinity maturation techniques) is likely to give rise to manymutants, most binding to antigen, and a few with higher affinity. Withlimiting TAT226, rare high affinity phage could be competed out. Toretain all higher affinity mutants, phages can be incubated with excessbiotinylated TAT226, but with the biotinylated TAT226 at a concentrationof lower molarity than the target molar affinity constant for TAT226.The high affinity-binding phages can then be captured bystreptavidin-coated paramagnetic beads. Such “equilibrium capture”allows the antibodies to be selected according to their affinities ofbinding, with sensitivity that permits isolation of mutant clones withas little as two-fold higher affinity from a great excess of phages withlower affinity. Conditions used in washing phages bound to a solid phasecan also be manipulated to discriminate on the basis of dissociationkinetics.

Anti-TAT226 clones may be selected based on activity. In certainembodiments, the invention provides anti-TAT226 antibodies that bind toliving cells that naturally express TAT226. In one embodiment, theinvention provides anti-TAT226 antibodies that block the binding betweena TAT226 ligand and TAT226, but do not block the binding between aTAT226 ligand and a second protein. Fv clones corresponding to suchanti-TAT226 antibodies can be selected by (1) isolating anti-TAT226clones from a phage library as described above, and optionallyamplifying the isolated population of phage clones by growing up thepopulation in a suitable bacterial host; (2) selecting TAT226 and asecond protein against which blocking and non-blocking activity,respectively, is desired; (3) adsorbing the anti-TAT226 phage clones toimmobilized TAT226; (4) using an excess of the second protein to eluteany undesired clones that recognize TAT226-binding determinants whichoverlap or are shared with the binding determinants of the secondprotein; and (5) eluting the clones which remain adsorbed following step(4). Optionally, clones with the desired blocking/non-blockingproperties can be further enriched by repeating the selection proceduresdescribed herein one or more times.

DNA encoding hybridoma-derived monoclonal antibodies or phage display Fvclones of the invention is readily isolated and sequenced usingconventional procedures (e.g. by using oligonucleotide primers designedto specifically amplify the heavy and light chain coding regions ofinterest from hybridoma or phage DNA template). Once isolated, the DNAcan be placed into expression vectors, which are then transfected intohost cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of the desiredmonoclonal antibodies in the recombinant host cells. Review articles onrecombinant expression in bacteria of antibody-encoding DNA includeSkerra et al., Curr. Opinion in Immunol., 5: 256 (1993) and Pluckthun,Immunol. Revs, 130: 151 (1992).

DNA encoding the Fv clones of the invention can be combined with knownDNA sequences encoding heavy chain and/or light chain constant regions(e.g. the appropriate DNA sequences can be obtained from Kabat et al.,supra) to form clones encoding full or partial length heavy and/or lightchains. It will be appreciated that constant regions of any isotype canbe used for this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species. An Fv clone derived from the variable domain DNA ofone animal (such as human) species and then fused to constant region DNAof another animal species to form coding sequence(s) for “hybrid,” fulllength heavy chain and/or light chain is included in the definition of“chimeric” and “hybrid” antibody as used herein. In certain embodiments,an Fv clone derived from human variable DNA is fused to human constantregion DNA to form coding sequence(s) for full- or partial-length humanheavy and/or light chains.

DNA encoding anti-TAT226 antibody derived from a hybridoma of theinvention can also be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofhomologous murine sequences derived from the hybridoma clone (e.g. as inthe method of Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855(1984)). DNA encoding a hybridoma- or Fv clone-derived antibody orfragment can be further modified by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In this manner, “chimeric” or “hybrid”antibodies are prepared that have the binding specificity of the Fvclone or hybridoma clone-derived antibodies of the invention.

3. Vectors, Host Cells, and Recombinant Methods

For recombinant production of an antibody of the invention, the nucleicacid encoding it is isolated and inserted into a replicable vector forfurther cloning (amplification of the DNA) or for expression. DNAencoding the antibody is readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors are available. The choice ofvector depends in part on the host cell to be used. Generally, hostcells are of either prokaryotic or eukaryotic (generally mammalian)origin. It will be appreciated that constant regions of any isotype canbe used for this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species.

a) Generating Antibodies Using Prokaryotic Host Cells:

(1) Vector Construction

Polynucleotide sequences encoding polypeptide components of the antibodyof the invention can be obtained using standard recombinant techniques.Desired polynucleotide sequences may be isolated and sequenced fromantibody producing cells such as hybridoma cells. Alternatively,polynucleotides can be synthesized using nucleotide synthesizer or PCRtechniques. Once obtained, sequences encoding the polypeptides areinserted into a recombinant vector capable of replicating and expressingheterologous polynucleotides in prokaryotic hosts. Many vectors that areavailable and known in the art can be used for the purpose of thepresent invention. Selection of an appropriate vector will depend mainlyon the size of the nucleic acids to be inserted into the vector and theparticular host cell to be transformed with the vector. Each vectorcontains various components, depending on its function (amplification orexpression of heterologous polynucleotide, or both) and itscompatibility with the particular host cell in which it resides. Thevector components generally include, but are not limited to: an originof replication, a selection marker gene, a promoter, a ribosome bindingsite (RBS), a signal sequence, the heterologous nucleic acid insert anda transcription termination sequence.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies. pBR322 contains genes encoding ampicillin (Amp) andtetracycline (Tet) resistance and thus provides easy means foridentifying transformed cells. pBR322, its derivatives, or othermicrobial plasmids or bacteriophage may also contain, or be modified tocontain, promoters which can be used by the microbial organism forexpression of endogenous proteins. Examples of pBR322 derivatives usedfor expression of particular antibodies are described in detail inCarter et al., U.S. Pat. No. 5,648,237.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM.TM.-11 may be utilized in making arecombinant vector which can be used to transform susceptible host cellssuch as E. coli LE392.

The expression vector of the invention may comprise two or morepromoter-cistron pairs, encoding each of the polypeptide components. Apromoter is an untranslated regulatory sequence located upstream (5′) toa cistron that modulates its expression. Prokaryotic promoters typicallyfall into two classes, inducible and constitutive. Inducible promoter isa promoter that initiates increased levels of transcription of thecistron under its control in response to changes in the culturecondition, e.g. the presence or absence of a nutrient or a change intemperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding the light or heavy chain by removing the promoterfrom the source DNA via restriction enzyme digestion and inserting theisolated promoter sequence into the vector of the invention. Both thenative promoter sequence and many heterologous promoters may be used todirect amplification and/or expression of the target genes. In someembodiments, heterologous promoters are utilized, as they generallypermit greater transcription and higher yields of expressed target geneas compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoApromoter, the β-galactamase and lactose promoter systems, a tryptophan(trp) promoter system and hybrid promoters such as the tac or the trcpromoter. However, other promoters that are functional in bacteria (suchas other known bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledworker operably to ligate them to cistrons encoding the target light andheavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers oradaptors to supply any required restriction sites.

In one aspect of the invention, each cistron within the recombinantvector comprises a secretion signal sequence component that directstranslocation of the expressed polypeptides across a membrane. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the target polypeptide DNA that is inserted into thevector. The signal sequence selected for the purpose of this inventionshould be one that is recognized and processed (i.e. cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the signal sequences native to the heterologouspolypeptides, the signal sequence is substituted by a prokaryotic signalsequence selected, for example, from the group consisting of thealkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II(STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of theinvention, the signal sequences used in both cistrons of the expressionsystem are STII signal sequences or variants thereof.

In another aspect, the production of the immunoglobulins according tothe invention can occur in the cytoplasm of the host cell, and thereforedoes not require the presence of secretion signal sequences within eachcistron. In that regard, immunoglobulin light and heavy chains areexpressed, folded and assembled to form functional immunoglobulinswithin the cytoplasm. Certain host strains (e.g., the E. colitrxB-strains) provide cytoplasm conditions that are favorable fordisulfide bond formation, thereby permitting proper folding and assemblyof expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

Antibodies of the invention can also be produced by using an expressionsystem in which the quantitative ratio of expressed polypeptidecomponents can be modulated in order to maximize the yield of secretedand properly assembled antibodies of the invention. Such modulation isaccomplished at least in part by simultaneously modulating translationalstrengths for the polypeptide components.

One technique for modulating translational strength is disclosed inSimmons et al., U.S. Pat. No. 5,840,523. It utilizes variants of thetranslational initiation region (TIR) within a cistron. For a given TIR,a series of amino acid or nucleic acid sequence variants can be createdwith a range of translational strengths, thereby providing a convenientmeans by which to adjust this factor for the desired expression level ofthe specific chain. TIR variants can be generated by conventionalmutagenesis techniques that result in codon changes which can alter theamino acid sequence. In certain embodiments, changes in the nucleotidesequence are silent. Alterations in the TIR can include, for example,alterations in the number or spacing of Shine-Dalgarno sequences, alongwith alterations in the signal sequence. One method for generatingmutant signal sequences is the generation of a “codon bank” at thebeginning of a coding sequence that does not change the amino acidsequence of the signal sequence (i.e., the changes are silent). This canbe accomplished by changing the third nucleotide position of each codon;additionally, some amino acids, such as leucine, serine, and arginine,have multiple first and second positions that can add complexity inmaking the bank. This method of mutagenesis is described in detail inYansura et al. (1992) METHODS: A Companion to Methods in Enzymol.4:151-158.

In one embodiment, a set of vectors is generated with a range of TIRstrengths for each cistron therein. This limited set provides acomparison of expression levels of each chain as well as the yield ofthe desired antibody products under various TIR strength combinations.TIR strengths can be determined by quantifying the expression level of areporter gene as described in detail in Simmons et al. U.S. Pat. No.5,840,523. Based on the translational strength comparison, the desiredindividual TIRs are selected to be combined in the expression vectorconstructs of the invention.

Prokaryotic host cells suitable for expressing antibodies of theinvention include Archaebacteria and Eubacteria, such as Gram-negativeor Gram-positive organisms. Examples of useful bacteria includeEscherichia (e.g., E. coli), Bacilli (e.g., B. subtilis),Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonellatyphimurium, Serratia marcescans, Klebsiella, Proteus, Shigella,Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, gram-negativecells are used. In one embodiment, E. coli cells are used as hosts forthe invention. Examples of E. coli strains include strain W3110(Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.:American Society for Microbiology, 1987), pp. 1190-1219; ATCC DepositNo. 27,325) and derivatives thereof, including strain 33D3 havinggenotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE) degP41kanR (U.S. Pat. No. 5,639,635). Other strains and derivatives thereof,such as E. coli 294 (ATCC 31,446), E. coli B, E. coli λ 1776 (ATCC31,537) and E. coli RV308 (ATCC 31,608) are also suitable. Theseexamples are illustrative rather than limiting. Methods for constructingderivatives of any of the above-mentioned bacteria having definedgenotypes are known in the art and described in, for example, Bass etal., Proteins, 8:309-314 (1990). It is generally necessary to select theappropriate bacteria taking into consideration replicability of thereplicon in the cells of a bacterium. For example, E. coli, Serratia, orSalmonella species can be suitably used as the host when well knownplasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supplythe replicon. Typically the host cell should secrete minimal amounts ofproteolytic enzymes, and additional protease inhibitors may desirably beincorporated in the cell culture.

(2) Antibody Production

Host cells are transformed with the above-described expression vectorsand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation.

Prokaryotic cells used to produce the polypeptides of the invention aregrown in media known in the art and suitable for culture of the selectedhost cells. Examples of suitable media include luria broth (LB) plusnecessary nutrient supplements. In some embodiments, the media alsocontains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. Incertain embodiments, for E. coli growth, growth temperatures range fromabout 20° C. to about 39° C.; from about 25° C. to about 37° C.; orabout 30° C. The pH of the medium may be any pH ranging from about 5 toabout 9, depending mainly on the host organism. In certain embodiments,for E. coli, the pH is from about 6.8 to about 7.4, or about 7.0.

If an inducible promoter is used in the expression vector of theinvention, protein expression is induced under conditions suitable forthe activation of the promoter. In one aspect of the invention, PhoApromoters are used for controlling transcription of the polypeptides.Accordingly, the transformed host cells are cultured in aphosphate-limiting medium for induction. In certain embodiments, thephosphate-limiting medium is the C.R.A.P. medium (see, e.g., Simmons etal., J. Immunol. Methods (2002), 263:133-147). A variety of otherinducers may be used, according to the vector construct employed, as isknown in the art.

In one embodiment, the expressed polypeptides of the present inventionare secreted into and recovered from the periplasm of the host cells.Protein recovery typically involves disrupting the microorganism,generally by such means as osmotic shock, sonication or lysis. Oncecells are disrupted, cell debris or whole cells may be removed bycentrifugation or filtration. The proteins may be further purified, forexample, by affinity resin chromatography. Alternatively, proteins canbe transported into the culture media and isolated therein. Cells may beremoved from the culture and the culture supernatant being filtered andconcentrated for further purification of the proteins produced. Theexpressed polypeptides can be further isolated and identified usingcommonly known methods such as polyacrylamide gel electrophoresis (PAGE)and Western blot assay.

In one aspect of the invention, antibody production is conducted inlarge quantity by a fermentation process. Various large-scale fed-batchfermentation procedures are available for production of recombinantproteins. Large-scale fermentations have at least 1000 liters ofcapacity, and in certain embodiments, about 1,000 to 100,000 liters ofcapacity. These fermentors use agitator impellers to distribute oxygenand nutrients, especially glucose (the preferred carbon/energy source).Small scale fermentation refers generally to fermentation in a fermentorthat is no more than approximately 100 liters in volumetric capacity,and can range from about 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD550 of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the polypeptides of theinvention, various fermentation conditions can be modified. For example,to improve the proper assembly and folding of the secreted antibodypolypeptides, additional vectors overexpressing chaperone proteins, suchas Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (apeptidylprolyl cis,trans-isomerase with chaperone activity) can be usedto co-transform the host prokaryotic cells. The chaperone proteins havebeen demonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells. Chen et al.(1999) J. Biol. Chem. 274:19601-19605; Georgiou et al., U.S. Pat. No.6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann andPluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun(2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol.Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the present invention.For example, host cell strains may be modified to effect geneticmutation(s) in the genes encoding known bacterial proteases such asProtease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI and combinations thereof. Some E. coli protease-deficientstrains are available and described in, for example, Joly et al. (1998),supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S.Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72(1996).

In one embodiment, E. coli strains deficient for proteolytic enzymes andtransformed with plasmids overexpressing one or more chaperone proteinsare used as host cells in the expression system of the invention.

(3) Antibody Purification

In one embodiment, the antibody protein produced herein is furtherpurified to obtain preparations that are substantially homogeneous forfurther assays and uses. Standard protein purification methods known inthe art can be employed. The following procedures are exemplary ofsuitable purification procedures: fractionation on immunoaffinity orion-exchange columns, ethanol precipitation, reverse phase HPLC,chromatography on silica or on a cation-exchange resin such as DEAE,chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gelfiltration using, for example, Sephadex G-75.

In one aspect, Protein A immobilized on a solid phase is used forimmunoaffinity purification of the antibody products of the invention.Protein A is a 41 kD cell wall protein from Staphylococcus aureas whichbinds with a high affinity to the Fc region of antibodies. Lindmark etal (1983) J. Immunol. Meth. 62:1-13. The solid phase to which Protein Ais immobilized can be a column comprising a glass or silica surface, ora controlled pore glass column or a silicic acid column. In someapplications, the column is coated with a reagent, such as glycerol, topossibly prevent nonspecific adherence of contaminants.

As the first step of purification, a preparation derived from the cellculture as described above can be applied onto a Protein A immobilizedsolid phase to allow specific binding of the antibody of interest toProtein A. The solid phase would then be washed to remove contaminantsnon-specifically bound to the solid phase. Finally the antibody ofinterest is recovered from the solid phase by elution.

b) Generating Antibodies Using Eukaryotic Host Cells:

A vector for use in a eukaryotic host cell generally includes one ormore of the following non-limiting components: a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence.

(1) Signal Sequence Component

A vector for use in a eukaryotic host cell may also contain a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide of interest. Theheterologous signal sequence selected may be one that is recognized andprocessed (i.e., cleaved by a signal peptidase) by the host cell. Inmammalian cell expression, mammalian signal sequences as well as viralsecretory leaders, for example, the herpes simplex gD signal, areavailable. The DNA for such a precursor region is ligated in readingframe to DNA encoding the antibody.

(2) Origin of Replication

Generally, an origin of replication component is not needed formammalian expression vectors. For example, the SV40 origin may typicallybe used only because it contains the early promoter.

(3) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, where relevant, or (c) supply critical nutrients notavailable from complex media.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-Iand -II, preferably primate metallothionein genes, adenosine deaminase,ornithine decarboxylase, etc.

For example, in some embodiments, cells transformed with the DHFRselection gene are first identified by culturing all of thetransformants in a culture medium that contains methotrexate (Mtx), acompetitive antagonist of DHFR. In some embodiments, an appropriate hostcell when wild-type DHFR is employed is the Chinese hamster ovary (CHO)cell line deficient in DHFR activity (e.g., ATCC CRL-9096).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

(4) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to nucleic acidencoding a polypeptide of interest (e.g., an antibody). Promotersequences are known for eukaryotes. For example, virtually alleukaryotic genes have an AT-rich region located approximately 25 to 30bases upstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. In certain embodiments, any or all of these sequences may besuitably inserted into eukaryotic expression vectors.

Transcription from vectors in mammalian host cells is controlled, forexample, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovinepapilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus,hepatitis-B virus and Simian Virus 40 (SV40), from heterologousmammalian promoters, e.g., the actin promoter or an immunoglobulinpromoter, from heat-shock promoters, provided such promoters arecompatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982), describingexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

(5) Enhancer Element Component

Transcription of DNA encoding an antibody of this invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, α-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv, Nature 297:17-18 (1982) describing enhancerelements for activation of eukaryotic promoters. The enhancer may bespliced into the vector at a position 5′ or 3′ to the antibodypolypeptide-encoding sequence, but is generally located at a site 5′from the promoter.

(6) Transcription Termination Component

Expression vectors used in eukaryotic host cells may also containsequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from the 5′and, occasionally 3′, untranslated regions of eukaryotic or viral DNAsor cDNAs. These regions contain nucleotide segments transcribed aspolyadenylated fragments in the untranslated portion of the mRNAencoding an antibody. One useful transcription termination component isthe bovine growth hormone polyadenylation region. See WO94/11026 and theexpression vector disclosed therein.

(7) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein include higher eukaryote cells described herein, includingvertebrate host cells. Propagation of vertebrate cells in culture(tissue culture) has become a routine procedure. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinesehamster ovary cells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci.USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

(8) Culturing the Host Cells

The host cells used to produce an antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othersupplements may also be included at appropriate concentrations thatwould be known to those skilled in the art. The culture conditions, suchas temperature, pH, and the like, are those previously used with thehost cell selected for expression, and will be apparent to theordinarily skilled artisan.

(9) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, or directly secreted into the medium. If the antibodyis produced intracellularly, as a first step, the particulate debris,either host cells or lysed fragments, may be removed, for example, bycentrifugation or ultrafiltration. Where the antibody is secreted intothe medium, supernatants from such expression systems may be firstconcentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis, and antibiotics may be includedto prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing a convenient technique. The suitability of protein A as anaffinity ligand depends on the species and isotype of any immunoglobulinFc domain that is present in the antibody. Protein A can be used topurify antibodies that are based on human γ1, γ2, or γ4 heavy chains(Lindmark et al., J. Immunol. Methods 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached may be agarose, but other matrices are available. Mechanicallystable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to furtherpurification, for example, by low pH hydrophobic interactionchromatography using an elution buffer at a pH between about 2.5-4.5,preferably performed at low salt concentrations (e.g., from about0-0.25M salt).

In general, various methodologies for preparing antibodies for use inresearch, testing, and clinical use are well-established in the art,consistent with the above-described methodologies and/or as deemedappropriate by one skilled in the art for a particular antibody ofinterest.

C. Immunoconjugates

The invention also provides immunoconjugates (interchangeably referredto as “antibody-drug conjugates,” or “ADCs”) comprising any of theanti-TAT226 antibodies of the invention conjugated to one or morecytotoxic agents, such as a chemotherapeutic agent, a drug, a growthinhibitory agent, a toxin (e.g., an enzymatically active toxin ofbacterial, fungal, plant, or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate).

Immunoconjugates may be used for the local delivery of cytotoxic agents,i.e., drugs that kill or inhibit the growth or proliferation of tumorcells, in the treatment of cancer (Syrigos and Epenetos (1999)Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv.Drug Deliv. Rev. 26:151-172; U.S. Pat. No. 4,975,278) Immunoconjugatesallow for the targeted delivery of a drug moiety to a tumor, andintracellular accumulation therein, where systemic administration ofunconjugated drugs may result in unacceptable levels of toxicity tonormal cells as well as the tumor cells sought to be eliminated (Baldwinet al., Lancet (Mar. 15, 1986) pp. 603-05; Thorpe (1985) “AntibodyCarriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in MonoclonalAntibodies '84: Biological And Clinical Applications (A. Pinchera etal., eds) pp. 475-506. Both polyclonal antibodies and monoclonalantibodies have been reported as useful in these strategies (Rowland etal., (1986) Cancer Immunol. Immunother. 21:183-87). Drugs used in thesemethods include daunomycin, doxorubicin, methotrexate, and vindesine(Rowland et al., (1986) supra). Toxins used in antibody-toxin conjugatesinclude bacterial toxins such as diphtheria toxin, plant toxins such asricin, small molecule toxins such as geldanamycin (Mandler et al (2000)J. of the Nat. Cancer Inst. 92(19):1573-1581; Mandler et al (2000)Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al (2002)Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al.,(1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lodeet al (1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res.53:3336-3342). The toxins may exert their cytotoxic effects bymechanisms including tubulin binding, DNA binding, or topoisomeraseinhibition. Some cytotoxic drugs tend to be inactive or less active whenconjugated to large antibodies or protein receptor ligands.

ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotopeconjugate composed of a murine IgG1 kappa monoclonal antibody directedagainst the CD20 antigen found on the surface of normal and malignant Blymphocytes and 111In or 90Y radioisotope bound by a thiourealinker-chelator (Wiseman et al (2000) Eur. Jour. Nucl. Med.27(7):766-77; Wiseman et al (2002) Blood 99(12):4336-42; Witzig et al(2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al (2002) J. Clin.Oncol. 20(15):3262-69). Although ZEVALIN has activity against B-cellnon-Hodgkin's Lymphoma (NHL), administration results in severe andprolonged cytopenias in most patients. MYLOTARG™ (gemtuzumab ozogamicin,Wyeth Pharmaceuticals), an antibody-drug conjugate composed of a hu CD33antibody linked to calicheamicin, was approved in 2000 for the treatmentof acute myeloid leukemia by injection (Drugs of the Future (2000)25(7):686; U.S. Pat. Nos. 4,970,198; 5,079,233; 5,585,089; 5,606,040;5,693,762; 5,739,116; 5,767,285; 5,773,001). Cantuzumab mertansine(Immunogen, Inc.), an antibody-drug conjugate composed of the huC242antibody linked via the disulfide linker SPP to the maytansinoid drugmoiety, DM1, is advancing into Phase II trials for the treatment ofcancers that express CanAg, such as colon, pancreatic, gastric, andothers. MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), anantibody-drug conjugate composed of the anti-prostate specific membraneantigen (PSMA) monoclonal antibody linked to the maytansinoid drugmoiety, DM1, is under development for the potential treatment ofprostate tumors. The auristatin peptides, auristatin E (AE) andmonomethylauristatin (MMAE), synthetic analogs of dolastatin, wereconjugated to chimeric monoclonal antibodies cBR96 (specific to Lewis Yon carcinomas) and cAC10 (specific to CD30 on hematologicalmalignancies) (Doronina et al (2003) Nature Biotechnol. 21(7):778-784)and are under therapeutic development.

In certain embodiments, an immunoconjugate comprises an anti-TAT226antibody and a chemotherapeutic agent or other toxin. Chemotherapeuticagents useful in the generation of immunoconjugates are described herein(e.g., above). Enzymatically active toxins and fragments thereof canalso be used and are described herein.

In certain embodiments, an immunoconjugate comprises an anti-TAT226antibody and one or more small molecule toxins, including, but notlimited to, small molecule drugs such as a calicheamicin, maytansinoid,dolastatin, auristatin, trichothecene, and CC1065, and the derivativesof these drugs that have cytotoxic activity. Examples of suchimmunoconjugates are discussed in further detail below.

1. Exemplary Immunoconjugates

An immunoconjugate (or “antibody-drug conjugate” (“ADC”)) of theinvention may be of Formula I, below, wherein an anti-TAT226 antibody isconjugated (i.e., covalently attached) to one or more drug moieties (D)through an optional linker (L).Ab-(L-D)_(p)  IAccordingly, the anti-TAT226 antibody may be conjugated to the drugeither directly or via a linker. In Formula I, p is the average numberof drug moieties per antibody, which can range, e.g., from about 1 toabout 20 drug moieties per antibody, and in certain embodiments, from 1to about 8 drug moieties per antibody.

a) Exemplary Linkers

A linker may comprise one or more linker components. Exemplary linkercomponents include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”),valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (a “PAB”), N-Succinimidyl4-(2-pyridylthio) pentanoate (“SPP”), N-succinimidyl4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“SMCC”), andN-Succinimidyl (4-iodo-acetyl) aminobenzoate (“SIAB”). Various linkercomponents are known in the art, some of which are described below.

A linker may be a “cleavable linker,” facilitating release of a drug inthe cell. For example, an acid-labile linker (e.g., hydrazone),protease-sensitive (e.g., peptidase-sensitive) linker, photolabilelinker, dimethyl linker or disulfide-containing linker (Chari et al.,Cancer Research 52:127-131 (1992); U.S. Pat. No. 5,208,020) may be used.

In some embodiments, a linker component may comprise a “stretcher unit”that links an antibody to another linker component or to a drug moiety.Exemplary stretcher units are shown below (wherein the wavy lineindicates sites of covalent attachment to an antibody):

In some embodiments, a linker component may comprise an amino acid unit.In one such embodiment, the amino acid unit allows for cleavage of thelinker by a protease, thereby facilitating release of the drug from theimmunoconjugate upon exposure to intracellular proteases, such aslysosomal enzymes. See, e.g., Doronina et al. (2003) Nat. Biotechnol.21:778-784. Exemplary amino acid units include, but are not limited to,a dipeptide, a tripeptide, a tetrapeptide, and a pentapeptide. Exemplarydipeptides include: valine-citrulline (vc or val-cit),alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk orphe-lys); or N-methyl-valine-citrulline (Me-val-cit). Exemplarytripeptides include: glycine-valine-citrulline (gly-val-cit) andglycine-glycine-glycine (gly-gly-gly). An amino acid unit may compriseamino acid residues that occur naturally, as well as minor amino acidsand non-naturally occurring amino acid analogs, such as citrulline Aminoacid units can be designed and optimized in their selectivity forenzymatic cleavage by a particular enzyme, for example, atumor-associated protease, cathepsin B, C and D, or a plasmin protease.

In some embodiments, a linker component may comprise a “spacer” unitthat links the antibody to a drug moiety, either directly or by way of astretcher unit and/or an amino acid unit. A spacer unit may be“self-immolative” or a “non-self-immolative.” A “non-self-immolative”spacer unit is one in which part or all of the spacer unit remains boundto the drug moiety upon enzymatic (e.g., proteolytic) cleavage of theADC. Examples of non-self-immolative spacer units include, but are notlimited to, a glycine spacer unit and a glycine-glycine spacer unit.Other combinations of peptidic spacers susceptible to sequence-specificenzymatic cleavage are also contemplated. For example, enzymaticcleavage of an ADC containing a glycine-glycine spacer unit by atumor-cell associated protease would result in release of aglycine-glycine-drug moiety from the remainder of the ADC. In one suchembodiment, the glycine-glycine-drug moiety is then subjected to aseparate hydrolysis step in the tumor cell, thus cleaving theglycine-glycine spacer unit from the drug moiety.

A “self-immolative” spacer unit allows for release of the drug moietywithout a separate hydrolysis step. In certain embodiments, a spacerunit of a linker comprises a p-aminobenzyl unit. In one such embodiment,a p-aminobenzyl alcohol is attached to an amino acid unit via an amidebond, and a carbamate, methylcarbamate, or carbonate is made between thebenzyl alcohol and a cytotoxic agent. See, e.g., Hamann et al. (2005)Expert Opin. Ther. Patents (2005) 15:1087-1103. In one embodiment, thespacer unit is p-aminobenzyloxycarbonyl (PAB). In certain embodiments,the phenylene portion of a p-amino benzyl unit is substituted with Qm,wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;and m is an integer ranging from 0-4. Examples of self-immolative spacerunits further include, but are not limited to, aromatic compounds thatare electronically similar to p-aminobenzyl alcohol (see, e.g., US2005/0256030 A1), such as 2-aminoimidazol-5-methanol derivatives (Hay etal. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho- orpara-aminobenzylacetals. Spacers can be used that undergo cyclizationupon amide bond hydrolysis, such as substituted and unsubstituted4-aminobutyric acid amides (Rodrigues et al., Chemistry Biology, 1995,2, 223); appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2]ring systems (Storm, et al., J. Amer. Chem. Soc., 1972, 94, 5815); and2-aminophenylpropionic acid amides (Amsberry, et al., J. Org. Chem.,1990, 55, 5867). Elimination of amine-containing drugs that aresubstituted at the a-position of glycine (Kingsbury, et al., J. Med.Chem., 1984, 27, 1447) are also examples of self-immolative spacersuseful in ADCs.

In one embodiment, a spacer unit is a branched bis(hydroxymethyl)styrene(BHMS) unit as depicted below, which can be used to incorporate andrelease multiple drugs.

wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;m is an integer ranging from 0-4; n is 0 or 1; and p ranges raging from1 to about 20.

A linker may comprise any one or more of the above linker components. Incertain embodiments, a linker is as shown in brackets in the followingADC Formula IIAb

[Aa-Ww-Yy]-D)_(p)  IIwherein A is a stretcher unit, and a is an integer from 0 to 1; W is anamino acid unit, and w is an integer from 0 to 12; Y is a spacer unit,and y is 0, 1, or 2; and Ab, D, and p are defined as above for FormulaI. Exemplary embodiments of such linkers are described in US2005-0238649 A1, which is expressly incorporated herein by reference.

Exemplary linker components and combinations thereof are shown below inthe context of ADCs of Formula II:

Linkers components, including stretcher, spacer, and amino acid units,may be synthesized by methods known in the art, such as those describedin US 2005-0238649 A1.

b) Exemplary Drug Moieties

(1) Maytansine and Maytansinoids

In some embodiments, an immunoconjugate comprises an antibody of theinvention conjugated to one or more maytansinoid molecules.Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533.

Maytansinoid drug moieties are attractive drug moieties in antibody-drugconjugates because they are: (i) relatively accessible to prepare byfermentation or chemical modification or derivatization of fermentationproducts, (ii) amenable to derivatization with functional groupssuitable for conjugation through non-disulfide linkers to antibodies,(iii) stable in plasma, and (iv) effective against a variety of tumorcell lines.

Maytansine compounds suitable for use as maytansinoid drug moieties arewell known in the art and can be isolated from natural sources accordingto known methods or produced using genetic engineering techniques (seeYu et al (2002) PNAS 99:7968-7973). Maytansinol and maytansinolanalogues may also be prepared synthetically according to known methods.

Exemplary maytansinoid drug moieties include those having a modifiedaromatic ring, such as: C-19-dechloro (U.S. Pat. No. 4,256,746)(prepared by lithium aluminum hydride reduction of ansamytocin P2);C-20-hydroxy (or C-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos.4,361,650 and 4,307,016) (prepared by demethylation using Streptomycesor Actinomyces or dechlorination using LAH); and C-20-demethoxy,C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No. 4,294,757) (prepared byacylation using acyl chlorides). and those having modifications at otherpositions.

Exemplary maytansinoid drug moieties also include those havingmodifications such as: C-9-SH (U.S. Pat. No. 4,424,219) (prepared by thereaction of maytansinol with H₂S or P₂S₅);C-14-alkoxymethyl(demethoxy/CH₂OR)(U.S. Pat. No. 4,331,598);C-14-hydroxymethyl or acyloxymethyl (CH₂OH or CH₂OAc) (U.S. Pat. No.4,450,254) (prepared from Nocardia); C-15-hydroxy/acyloxy (U.S. Pat. No.4,364,866) (prepared by the conversion of maytansinol by Streptomyces);C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated fromTrewia nudlflora); C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and4,322,348) (prepared by the demethylation of maytansinol byStreptomyces); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by thetitanium trichloride/LAH reduction of maytansinol).

Exemplary embodiments of maytansinoid drug moieties include: DM1; DM3;and DM4, having the structures:

wherein the wavy line indicates the covalent attachment of the sulfuratom of the drug to a linker (L) of an antibody-drug conjugate.HERCEPTIN® (trastuzumab) linked by SMCC to DM1 has been reported (WO2005/037992; US 2005/0276812 A1).

Other exemplary maytansinoid antibody-drug conjugates have the followingstructures and abbreviations, (wherein Ab is antibody and p is 1 toabout 8):

Exemplary antibody-drug conjugates where DM1 is linked through a BMPEOlinker to a thiol group of the antibody have the structure andabbreviation:

where Ab is antibody; n is 0, 1, or 2; and p is 1, 2, 3, or 4.

Immunoconjugates containing maytansinoids, methods of making the same,and their therapeutic use are disclosed, for example, in U.S. Pat. Nos.5,208,020, 5,416,064, US 2005/0276812 A1, and European Patent EP 0 425235 B1, the disclosures of which are hereby expressly incorporated byreference. Liu et al. Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996)describe immunoconjugates comprising a maytansinoid designated DM1linked to the monoclonal antibody C242 directed against human colorectalcancer. The conjugate was found to be highly cytotoxic towards culturedcolon cancer cells, and showed antitumor activity in an in vivo tumorgrowth assay. Chari et al. Cancer Research 52:127-131 (1992) describeimmunoconjugates in which a maytansinoid was conjugated via a disulfidelinker to the murine antibody A7 binding to an antigen on human coloncancer cell lines, or to another murine monoclonal antibody TA.1 thatbinds the HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansonoidconjugate was tested in vitro on the human breast cancer cell lineSK-BR-3, which expresses 3×10⁵ HER-2 surface antigens per cell. The drugconjugate achieved a degree of cytotoxicity similar to the freemaytansinoid drug, which could be increased by increasing the number ofmaytansinoid molecules per antibody molecule. The A7-maytansinoidconjugate showed low systemic cytotoxicity in mice.

Antibody-maytansinoid conjugates are prepared by chemically linking anantibody to a maytansinoid molecule without significantly diminishingthe biological activity of either the antibody or the maytansinoidmolecule. See, e.g., U.S. Pat. No. 5,208,020 (the disclosure of which ishereby expressly incorporated by reference). An average of 3-4maytansinoid molecules conjugated per antibody molecule has shownefficacy in enhancing cytotoxicity of target cells without negativelyaffecting the function or solubility of the antibody, although even onemolecule of toxin/antibody would be expected to enhance cytotoxicityover the use of naked antibody. Maytansinoids are well known in the artand can be synthesized by known techniques or isolated from naturalsources. Suitable maytansinoids are disclosed, for example, in U.S. Pat.No. 5,208,020 and in the other patents and nonpatent publicationsreferred to hereinabove. Preferred maytansinoids are maytansinol andmaytansinol analogues modified in the aromatic ring or at otherpositions of the maytansinol molecule, such as various maytansinolesters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1; Chari etal. Cancer Research 52:127-131 (1992); and US 2005/016993 A1, thedisclosures of which are hereby expressly incorporated by reference.Antibody-maytansinoid conjugates comprising the linker component SMCCmay be prepared as disclosed in US 2005/0276812 A1, “Antibody-drugconjugates and Methods.” The linkers comprise disulfide groups,thioether groups, acid labile groups, photolabile groups, peptidaselabile groups, or esterase labile groups, as disclosed in theabove-identified patents. Additional linkers are described andexemplified herein.

Conjugates of the antibody and maytansinoid may be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). In certain embodiments, the couplingagent is N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlssonet al., Biochem. J. 173:723-737 (1978)) orN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. In one embodiment, the linkage isformed at the C-3 position of maytansinol or a maytansinol analogue.

(2) Auristatins and Dolastatins

In some embodiments, an immunoconjugate comprises an antibody of theinvention conjugated to dolastatin or a dolastatin peptidic analog orderivative, e.g., an auristatin (U.S. Pat. Nos. 5,635,483; 5,780,588).Dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity(Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). Thedolastatin or auristatin drug moiety may be attached to the antibodythrough the N (amino) terminus or the C (carboxyl) terminus of thepeptidic drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in Senter et al,Proceedings of the American Association for Cancer Research, Volume 45,Abstract Number 623, presented Mar. 28, 2004, the disclosure of which isexpressly incorporated by reference in its entirety.

A peptidic drug moiety may be selected from Formulas D_(E) and D_(F)below:

wherein the wavy line of D_(E) and D_(F) indicates the covalentattachment site to an antibody or antibody-linker component, andindependently at each location:

R² is selected from H and C₁-C₈ alkyl;

R³ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle);

R⁴ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle);

R⁵ is selected from H and methyl;

or R⁴ and R⁵ jointly form a carbocyclic ring and have the formula—(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selectedfrom H, C₁-C₈ alkyl and C₃-C₈ carbocycle and n is selected from 2, 3, 4,5 and 6;

R⁶ is selected from H and C₁-C₈ alkyl;

R⁷ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle);

each R⁸ is independently selected from H, OH, C₁-C₈ alkyl, C₃-C₈carbocycle and O—(C₁-C₈ alkyl);

R⁹ is selected from H and C₁-C₈ alkyl;

R¹⁰ is selected from aryl or C₃-C₈ heterocycle;

Z is O, S, NH, or NR¹², wherein R¹² is C₁-C₈ alkyl;

R¹¹ is selected from H, C₁-C₂₀ alkyl, aryl, C₃-C₈ heterocycle,—(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—CH(R¹⁵)₂;

m is an integer ranging from 1-1000;

R¹³ is C₂-C₈ alkyl;

R¹⁴ is H or C₁-C₈ alkyl;

each occurrence of R¹⁵ is independently H, COOH, —(CH₂)_(n)—N(R¹⁶)₂,—(CH₂)_(n)—SO₃H, or —(CH₂)_(n)—SO₃—C₁-C₈ alkyl;

each occurrence of R¹⁶ is independently H, C₁-C₈ alkyl, or—(CH₂)_(n)—COOH;

R¹⁸ is selected from —C(R⁸)₂—C(R⁸)₂-aryl, —C(R⁸)₂—C(R⁸)₂—(C₃-C₈heterocycle), and —C(R⁸)₂—C(R⁸)₂—(C₃-C₈ carbocycle); and

n is an integer ranging from 0 to 6.

In one embodiment, R³, R⁴ and R⁷ are independently isopropyl orsec-butyl and R⁵ is —H or methyl. In an exemplary embodiment, R³ and R⁴are each isopropyl, R⁵ is —H, and R⁷ is sec-butyl.

In yet another embodiment, R² and R⁶ are each methyl, and R⁹ is —H.

In still another embodiment, each occurrence of R⁸ is —OCH₃.

In an exemplary embodiment, R³ and R⁴ are each isopropyl, R² and R⁶ areeach methyl, R⁵ is —H, R⁷ is sec-butyl, each occurrence of R⁸ is —OCH₃,and R⁹ is —H.

In one embodiment, Z is —O— or —NH—.

In one embodiment, R¹⁰ is aryl.

In an exemplary embodiment, R¹⁰ is -phenyl.

In an exemplary embodiment, when Z is —O—, R¹¹ is —H, methyl or t-butyl.

In one embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂)_(n)N(R¹⁶)₂, and R¹⁶ is —C₁-C₈ alkyl or —(CH₂)_(n)—COOH.

In another embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂)_(n)—SO₃H.

An exemplary auristatin embodiment of formula D_(E) is MMAE, wherein thewavy line indicates the covalent attachment to a linker (L) of anantibody-drug conjugate:

An exemplary auristatin embodiment of formula D_(F) is MMAF, wherein thewavy line indicates the covalent attachment to a linker (L) of anantibody-drug conjugate (see US 2005/0238649 and Doronina et al. (2006)Bioconjugate Chem. 17:114-124):

Other drug moieties include the following MMAF derivatives, wherein thewavy line indicates the covalent attachment to a linker (L) of anantibody-drug conjugate:

In one aspect, hydrophilic groups including but not limited to,triethylene glycol esters (TEG), as shown above, can be attached to thedrug moiety at R¹¹. Without being bound by any particular theory, thehydrophilic groups assist in the internalization and non-agglomerationof the drug moiety.

Exemplary embodiments of ADCs of Formula I comprising anauristatinldolastatin or derivative thereof are described in US2005-0238649 A1 and Doronina et al. (2006) Bioconjugate Chem.17:114-124, which is expressly incorporated herein by reference.Exemplary embodiments of ADCs of Formula I comprising MMAE or MMAF andvarious linker components have the following structures andabbreviations (wherein “Ab” is an antibody; p is 1 to about 8, “Val-Cit”is a valine-citrulline dipeptide; and “S” is a sulfur atom:

Exemplary embodiments of ADCs of Formula I comprising MMAF and variouslinker components further include Ab-MC-PAB-MMAF and Ab-PAB-MMAF.Interestingly, immunoconjugates comprising MMAF attached to an antibodyby a linker that is not proteolytically cleavable have been shown topossess activity comparable to immunoconjugates comprising MMAF attachedto an antibody by a proteolytically cleavable linker. See, Doronina etal. (2006) Bioconjugate Chem. 17:114-124. In such instances, drugrelease is believed to be effected by antibody degradation in the cell.Id.

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schröder and K. Liibke, “The Peptides”,volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. Auristatin/dolastatin drug moieties may beprepared according to the methods of: US 2005-0238649 A1; U.S. Pat. No.5,635,483; U.S. Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem.Soc. 111:5463-5465; Pettit et al (1998) Anti-Cancer Drug Design13:243-277; Pettit, G. R., et al. Synthesis, 1996, 719-725; Pettit et al(1996) J. Chem. Soc. Perkin Trans. 1 5:859-863; and Doronina (2003) Nat.Biotechnol. 21(7):778-784.

In particular, auristatin/dolastatin drug moieties of formula D_(F),such as MMAF and derivatives thereof, may be prepared using methodsdescribed in US 2005-0238649 A1 and Doronina et al. (2006) BioconjugateChem. 17:114-124. Auristatin/dolastatin drug moieties of formula D_(E),such as MMAE and derivatives thereof, may be prepared using methodsdescribed in Doronina et al. (2003) Nat. Biotech. 21:778-784.Drug-linker moieties MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF, andMC-vc-PAB-MMAE may be conveniently synthesized by routine methods, e.g.,as described in Doronina et al. (2003) Nat. Biotech. 21:778-784, andPatent Application Publication No. US 2005/0238649 A1, and thenconjugated to an antibody of interest.

(3) Calicheamicin

In other embodiments, the immunoconjugate comprises an antibody of theinvention conjugated to one or more calicheamicin molecules. Thecalicheamicin family of antibiotics are capable of producingdouble-stranded DNA breaks at sub-picomolar concentrations. For thepreparation of conjugates of the calicheamicin family, see U.S. Pat.Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710,5,773,001, 5,877,296 (all to American Cyanamid Company). Structuralanalogues of calicheamicin which may be used include, but are notlimited to, γ₁ ^(I), α₂ ^(I), α₃ ^(I), N-acetyl-γ₁ ^(I), PSAG and θ^(I)₁ (Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al.,Cancer Research 58:2925-2928 (1998), and the aforementioned U.S. patentsto American Cyanamid). Another anti-tumor drug to which the antibody canbe conjugated is QFA, which is an antifolate. Both calicheamicin and QFAhave intracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents throughantibody-mediated internalization greatly enhances their cytotoxiceffects.

c) Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodies of theinvention include BCNU, streptozocin, vincristine and 5-fluorouracil,the family of agents known collectively as the LL-E33288 complex,described in U.S. Pat. Nos. 5,053,394, 5,770,710, as well asesperamicins (U.S. Pat. No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

In certain embodiments, an immunoconjugate may comprise a highlyradioactive atom. A variety of radioactive isotopes are available forthe production of radioconjugated antibodies. Examples include At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactiveisotopes of Lu. When the immunoconjugate is used for detection, it maycomprise a radioactive atom for scintigraphic studies, for exampletc^(99m) or I¹²³, or a spin label for nuclear magnetic resonance (NMR)imaging (also known as magnetic resonance imaging, mri), such asiodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15,oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the immunoconjugate inknown ways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc^(99m) or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attachedvia a cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

In certain embodiments, an immunoconjugate may comprise an anti-TAT226antibody of the invention conjugated to a prodrug-activating enzyme thatconverts a prodrug (e.g., a peptidyl chemotherapeutic agent, see WO81/01145) to an active drug, such as an anti-cancer drug. Suchimmunoconjugates are useful in antibody-dependent enzyme-mediatedprodrug therapy (“ADEPT”). Enzymes that may be conjugated to ananti-TAT226 antibody of the invention include, but are not limited to,alkaline phosphatases, which are useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatases, which areuseful for converting sulfate-containing prodrugs into free drugs;cytosine deaminase, which is useful for converting non-toxic5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases,such as serratia protease, thermolysin, subtilisin, carboxypeptidasesand cathepsins (such as cathepsins B and L), which are useful forconverting peptide-containing prodrugs into free drugs;D-alanylcarboxypeptidases, which are useful for converting prodrugs thatcontain D-amino acid substituents; carbohydrate-cleaving enzymes such asβ-galactosidase and neuraminidase, which are useful for convertingglycosylated prodrugs into free drugs; β-lactamase, which is useful forconverting drugs derivatized with β-lactams into free drugs; andpenicillin amidases, such as penicillin V amidase and penicillin Gamidase, which are useful for converting drugs derivatized at theiramine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively,into free drugs. Enzymes may be be covalently bound to the anti-TAT226antibodies of the invention by recombinant DNA techniques well known inthe art. See, e.g., Neuberger et al., Nature 312:604-608 (1984).

d) Drug Loading

Drug loading is represented by p, the average number of drug moietiesper antibody in a molecule of Formula I. Drug loading may range from 1to 20 drug moieties (D) per antibody. ADCs of Formula I includecollections of antibodies conjugated with a range of drug moieties, from1 to 20. The average number of drug moieties per antibody inpreparations of ADC from conjugation reactions may be characterized byconventional means such as mass spectroscopy, ELISA assay, and HPLC. Thequantitative distribution of ADC in terms of p may also be determined.In some instances, separation, purification, and characterization ofhomogeneous ADC where p is a certain value from ADC with other drugloadings may be achieved by means such as reverse phase HPLC orelectrophoresis.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, as in the exemplary embodiments above, an antibody mayhave only one or several cysteine thiol groups, or may have only one orseveral sufficiently reactive thiol groups through which a linker may beattached. In certain embodiments, higher drug loading, e.g. p>5, maycause aggregation, insolubility, toxicity, or loss of cellularpermeability of certain antibody-drug conjugates. In certainembodiments, the drug loading for an ADC of the invention ranges from 1to about 8; from about 2 to about 6; or from about 3 to about 5. Indeed,it has been shown that for certain ADCs, the optimal ratio of drugmoieties per antibody may be less than 8, and may be about 2 to about 5.See US 2005-0238649 A1.

In certain embodiments, fewer than the theoretical maximum of drugmoieties are conjugated to an antibody during a conjugation reaction. Anantibody may contain, for example, lysine residues that do not reactwith the drug-linker intermediate or linker reagent, as discussed below.Generally, antibodies do not contain many free and reactive cysteinethiol groups which may be linked to a drug moiety; indeed most cysteinethiol residues in antibodies exist as disulfide bridges. In certainembodiments, an antibody may be reduced with a reducing agent such asdithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partialor total reducing conditions, to generate reactive cysteine thiolgroups. In certain embodiments, an antibody is subjected to denaturingconditions to reveal reactive nucleophilic groups such as lysine orcysteine.

The loading (drug/antibody ratio) of an ADC may be controlled indifferent ways, e.g., by: (i) limiting the molar excess of drug-linkerintermediate or linker reagent relative to antibody, (ii) limiting theconjugation reaction time or temperature, and (iii) partial or limitingreductive conditions for cysteine thiol modification.

It is to be understood that where more than one nucleophilic groupreacts with a drug-linker intermediate or linker reagent followed bydrug moiety reagent, then the resulting product is a mixture of ADCcompounds with a distribution of one or more drug moieties attached toan antibody. The average number of drugs per antibody may be calculatedfrom the mixture by a dual ELISA antibody assay, which is specific forantibody and specific for the drug. Individual ADC molecules may beidentified in the mixture by mass spectroscopy and separated by HPLC,e.g. hydrophobic interaction chromatography (see, e.g., Hamblett, K. J.,et al. “Effect of drug loading on the pharmacology, pharmacokinetics,and toxicity of an anti-CD30 antibody-drug conjugate,” Abstract No. 624,American Association for Cancer Research, 2004 Annual Meeting, Mar.27-31, 2004, Proceedings of the AACR, Volume 45, March 2004; Alley, S.C., et al. “Controlling the location of drug attachment in antibody-drugconjugates,” Abstract No. 627, American Association for Cancer Research,2004 Annual Meeting, Mar. 27-31, 2004, Proceedings of the AACR, Volume45, March 2004). In certain embodiments, a homogeneous ADC with a singleloading value may be isolated from the conjugation mixture byelectrophoresis or chromatography.

e) Certain Methods of Preparing Immunconjugates

An ADC of Formula I may be prepared by several routes employing organicchemistry reactions, conditions, and reagents known to those skilled inthe art, including: (1) reaction of a nucleophilic group of an antibodywith a bivalent linker reagent to form Ab-L via a covalent bond,followed by reaction with a drug moiety D; and (2) reaction of anucleophilic group of a drug moiety with a bivalent linker reagent, toform D-L, via a covalent bond, followed by reaction with a nucleophilicgroup of an antibody. Exemplary methods for preparing an ADC of FormulaI via the latter route are described in US 2005-0238649 A1, which isexpressly incorporated herein by reference.

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol) or tricarbonylethylphosphine (TCEP), such that theantibody is fully or partially reduced. Each cysteine bridge will thusform, theoretically, two reactive thiol nucleophiles. Additionalnucleophilic groups can be introduced into antibodies throughmodification of lysine residues, e.g., by reacting lysine residues with2-iminothiolane (Traut's reagent), resulting in conversion of an amineinto a thiol. Reactive thiol groups may be introduced into an antibodyby introducing one, two, three, four, or more cysteine residues (e.g.,by preparing variant antibodies comprising one or more non-nativecysteine amino acid residues).

Antibody-drug conjugates of the invention may also be produced byreaction between an electrophilic group on an antibody, such as analdehyde or ketone carbonyl group, with a nucleophilic group on a linkerreagent or drug. Useful nucleophilic groups on a linker reagent include,but are not limited to, hydrazide, oxime, amino, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In oneembodiment, an antibody is modified to introduce electrophilic moietiesthat are capable of reacting with nucleophilic subsituents on the linkerreagent or drug. In another embodiment, the sugars of glycosylatedantibodies may be oxidized, e.g. with periodate oxidizing reagents, toform aldehyde or ketone groups which may react with the amine group oflinker reagents or drug moieties. The resulting imine Schiff base groupsmay form a stable linkage, or may be reduced, e.g. by borohydridereagents to form stable amine linkages. In one embodiment, reaction ofthe carbohydrate portion of a glycosylated antibody with eithergalactose oxidase or sodium meta-periodate may yield carbonyl (aldehydeand ketone) groups in the antibody that can react with appropriategroups on the drug (Hermanson, Bioconjugate Techniques). In anotherembodiment, antibodies containing N-terminal serine or threonineresidues can react with sodium meta-periodate, resulting in productionof an aldehyde in place of the first amino acid (Geoghegan & Stroh,(1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852). Such analdehyde can be reacted with a drug moiety or linker nucleophile.

Nucleophilic groups on a drug moiety include, but are not limited toamine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone,hydrazine carboxylate, and arylhydrazide groups capable of reacting toform covalent bonds with electrophilic groups on linker moieties andlinker reagents including: (i) active esters such as NHS esters, HOBtesters, haloformates, and acid halides; (ii) alkyl and benzyl halidessuch as haloacetamides; (iii) aldehydes, ketones, carboxyl, andmaleimide groups.

The compounds of the invention expressly contemplate, but are notlimited to, ADC prepared with the following cross-linker reagents: BMPS,EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH,sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC,and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) whichare commercially available (e.g., from Pierce Biotechnology, Inc.,Rockford, Ill., U.S.A; see pages 467-498, 2003-2004 ApplicationsHandbook and Catalog.

Immunoconjugates comprising an antibody and a cytotoxic agent may alsobe made using a variety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

Alternatively, a fusion protein comprising an antibody and a cytotoxicagent may be made, e.g., by recombinant techniques or peptide synthesis.A recombinant DNA molecule may comprise regions encoding the antibodyand cytotoxic portions of the conjugate either adjacent to one anotheror separated by a region encoding a linker peptide which does notdestroy the desired properties of the conjugate.

In yet another embodiment, an antibody may be conjugated to a “receptor”(such as streptavidin) for utilization in tumor pre-targeting whereinthe antibody-receptor conjugate is administered to the patient, followedby removal of unbound conjugate from the circulation using a clearingagent and then administration of a “ligand” (e.g., avidin) which isconjugated to a cytotoxic agent (e.g., a radionucleotide).

D. Pharmaceutical Formulations

In one aspect, the invention further provides pharmaceuticalformulations comprising at least one anti-TAT226 antibody of theinvention and/or at least one immunoconjugate thereof. In someembodiments, a pharmaceutical formulation comprises 1) an anti-TAT226antibody and/or an immunoconjugate thereof, and 2) a pharmaceuticallyacceptable carrier. In some embodiments, a pharmaceutical formulationcomprises 1) an anti-TAT226 antibody and/or an immunoconjugate thereof,and optionally, 2) at least one additional therapeutic agent. Additionaltherapeutic agents include, but are not limited to, those describedbelow in Section E.2.

Pharmaceutical formulations comprising an antibody or immunoconjugate ofthe invention are prepared for storage by mixing the antibody orimmunoconjugate having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980))in the form of aqueous solutions or lyophilized or other driedformulations. Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, histidine and other organicacids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride);phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG). Pharmaceutical formulations to be used for invivo administration are generally sterile. This is readily accomplishedby filtration through sterile filtration membranes.

Active ingredients may also be entrapped in microcapsule prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsule andpoly-(methylmethacylate) microcapsule, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody or immunoconjugate of theinvention, which matrices are in the form of shaped articles, e.g.,films, or microcapsule. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate),or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919),copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated antibodies or immunoconjugates remain in thebody for a long time, they may denature or aggregate as a result ofexposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

E. Methods of Using Anti-TAT226 Antibodies and Immunoconjugates

1. Diagnostic Methods and Methods of Detection

In one aspect, anti-TAT226 antibodies and immunoconjugates of theinvention are useful for detecting the presence of TAT226 in abiological sample. The term “detecting” as used herein encompassesquantitative or qualitative detection. In certain embodiments, abiological sample comprises a cell or tissue, such as the tissues listedin FIG. 13. In certain embodiments, such tissues include normal and/orcancerous tissues that express TAT226 at higher levels relative to othertissues, for example, ovarian, kidney, brain, endometrial, adrenal,bone, lung, skin, and soft tissue.

In one aspect, the invention provides a method of detecting the presenceof TAT226 in a biological sample. In certain embodiments, the methodcomprises contacting the biological sample with an anti-TAT226 antibodyunder conditions permissive for binding of the anti-TAT226 antibody toTAT226, and detecting whether a complex is formed between theanti-TAT226 antibody and TAT226.

In one aspect, the invention provides a method of diagnosing a disorderassociated with increased expression of TAT226. In certain embodiments,the method comprises contacting a test cell with an anti-TAT226antibody; determining the level of expression (either quantitatively orqualitatively) of TAT226 by the test cell by detecting binding of theanti-TAT226 antibody to TAT226; and comparing the level of expression ofTAT226 by the test cell with the level of expression of TAT226 by acontrol cell (e.g., a normal cell of the same tissue origin as the testcell or a cell that expresses TAT226 at levels comparable to such anormal cell), wherein a higher level of expression of TAT226 by the testcell as compared to the control cell indicates the presence of adisorder associated with increased expression of TAT226. In certainembodiments, the test cell is obtained from an individual suspected ofhaving a disorder associated with increased expression of TAT226. Incertain embodiments, the disorder is a cell proliferative disorder, suchas a cancer or a tumor.

Exemplary cell proliferative disorders that may be diagnosed using anantibody of the invention include cancerous conditions such as tumors,e.g., carcinomas (epithelial tumors) and blastomas (embryonictissue-derived tumors), and in certain embodiments, ovarian cancer,uterine cancer, (including endometrial cancer), and kidney cancer,including nephroblastomas (e.g., Wilms' tumor). Ovarian cancer, inparticular, encompasses a heterogeneous group of malignant tumorsderived from the ovary. Approximately 90% of malignant ovarian tumorsare epithelial in origin; the remainder are germ cell and stromaltumors. Epithelial ovarian tumors are classified into the followinghistological subtypes: serous adenocarcinomas (constituting about 50% ofepithelial ovarian tumors); endometrioid adenocarcinomas (˜20%);mucinous adenocarcinomas (˜10%); clear cell carcinomas (˜5-10%); Brenner(transitional cell) tumors (relatively uncommon). The prognosis forovarian cancer, which is the sixth most common cancer in women, isusually poor, with five year survival rates ranging from 5-30%. Forreviews of ovarian cancer, see Fox et al. (2002) “Pathology ofepithelial ovarian cancer,” in Ovarian Cancer ch. 9 (Jacobs et al.,eds., Oxford University Press, New York); Morin et al. (2001) “OvarianCancer,” in Encyclopedic Reference of Cancer, pp. 654-656 (Schwab, ed.,Springer-Verlag, New York). The present invention contemplates methodsof diagnosing or treating any of the epithelial ovarian tumor subtypesdescribed above, and in particular, the serous adenocarcinoma subtype.

In certain embodiments, a method of diagnosis or detection, such asthose described above, comprises detecting binding of an anti-TAT226antibody to TAT226 expressed on the surface of a cell or in a membranepreparation obtained from a cell expressing TAT226 on its surface. Incertain embodiments, the method comprises contacting a cell with ananti-TAT226 antibody under conditions permissive for binding of theanti-TAT226 antibody to TAT226, and detecting whether a complex isformed between the anti-TAT226 antibody and TAT226 on the cell surface.An exemplary assay for detecting binding of an anti-TAT226 antibody toTAT226 expressed TAT226 on the surface of a cell is a “FACS” assay, suchas that described in Example D, below.

Certain other methods can be used to detect binding of anti-TAT226antibodies to TAT226. Such methods include, but are not limited to,antigen-binding assays that are well known in the art, such as westernblots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay),“sandwich” immunoassays, immunoprecipitation assays, fluorescentimmunoassays, protein A immunoassays, and immunohistochemistry (IHC).

In certain embodiments, anti-TAT226 antibodies are labeled. Labelsinclude, but are not limited to, labels or moieties that are detecteddirectly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

In certain embodiments, anti-TAT226 antibodies are immobilized on aninsoluble matrix. Immobilization entails separating the anti-TAT226antibody from any TAT226 that remains free in solution. Thisconventionally is accomplished by either insolubilizing the anti-TAT226antibody before the assay procedure, as by adsorption to awater-insoluble matrix or surface (Bennich et al., U.S. Pat. No.3,720,760), or by covalent coupling (for example, using glutaraldehydecross-linking), or by insolubilizing the anti-TAT226 antibody afterformation of a complex between the anti-TAT226 antibody and TAT226,e.g., by immunoprecipitation.

Any of the above embodiments of diagnosis or detection may be carriedout using an immunoconjugate of the invention in place of or in additionto an anti-TAT226 antibody.

2. Therapeutic Methods

An antibody or immunoconjugate of the invention may be used in, forexample, in vitro, ex vivo, and in vivo therapeutic methods. In oneaspect, the invention provides methods for inhibiting cell growth orproliferation, either in vivo or in vitro, the method comprisingexposing a cell to an anti-TAT226 antibody or immunoconjugate thereofunder conditions permissive for binding of the immunoconjugate to TAT226“Inhibiting cell growth or proliferation” means decreasing a cell'sgrowth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, or 100%, and includes inducing cell death. In certainembodiments, the cell is a tumor cell. In certain embodiments, the cellis an ovarian tumor cell, a uterine tumor cell, a brain tumor cell, or akidney tumor cell. In certain embodiments, the cell is a xenograft,e.g., as exemplified herein.

In one aspect, an antibody or immunoconjugate of the invention is usedto treat or prevent a cell proliferative disorder. In certainembodiments, the cell proliferative disorder is associated withincreased expression and/or activity of TAT226. For example, in certainembodiments, the cell proliferative disorder is associated withincreased expression of TAT226 on the surface of a cell. In certainembodiments, the cell proliferative disorder is a tumor or a cancer.Examples of cell proliferative disorders to be treated by the antibodiesor immunoconjugates of the invention include, but are not limited to,cancerous conditions such as tumors, e.g., carcinomas (epithelialtumors) and blastomas (embryonic tissue-derived tumors), and in certainembodiments, ovarian cancer; uterine cancer, including endometrialcancer; brain tumors (e.g., astrocytomas, encompassing advanced stagegliomas, also referred to as glioblastoma multiforme); and kidneycancer, including nephroblastomas (e.g., Wilms' tumor).

In one aspect, the invention provides methods for treating a cellproliferative disorder comprising administering to an individual aneffective amount of an anti-TAT226 antibody or immunoconjugate thereof.In certain embodiments, a method for treating a cell proliferativedisorder comprises administering to an individual an effective amount ofa pharmaceutical formulation comprising an anti-TAT226 antibody and,optionally, at least one additional therapeutic agent, such as thoseprovided below. In certain embodiments, a method for treating a cellproliferative disorder comprises administering to an individual aneffective amount of a pharmaceutical formulation comprising 1) animmunoconjugate comprising an anti-TAT226 antibody and a cytotoxicagent; and optionally, 2) at least one additional therapeutic agent,such as those provided below.

In one aspect, at least some of the antibodies or immunoconjugates ofthe invention can bind TAT226 from species other than human.Accordingly, antibodies or immunoconjugates of the invention can be usedto bind TAT226, e.g., in a cell culture containing TAT226, in humans, orin other mammals having a TAT226 with which an antibody orimmunoconjugate of the invention cross-reacts (e.g. chimpanzee, baboon,marmoset, cynomolgus and rhesus monkeys, pig or mouse). In oneembodiment, an anti-TAT226 antibody or immunoconjugate can be used forinhibiting a TAT226 activity by contacting the antibody orimmunoconjugate with TAT226 such that TAT226 activity is inhibited. Inone embodiment, the TAT226 is human TAT226.

In one embodiment, an anti-TAT226 antibody or immunoconjugate can beused in a method for binding TAT226 in an individual suffering from adisorder associated with increased TAT226 expression and/or activity,the method comprising administering to the individual the antibody orimmunoconjugate such that TAT226 in the individual is bound. In oneembodiment, the TAT226 is human TAT226, and the individual is a humanindividual. Alternatively, the individual can be a mammal expressingTAT226 to which an anti-TAT226 antibody binds. Still further theindividual can be a mammal into which TAT226 has been introduced (e.g.,by administration of TAT226 or by expression of a transgene encodingTAT226).

An anti-TAT226 antibody or immunoconjugate can be administered to ahuman for therapeutic purposes. Moreover, an anti-TAT226 antibody orimmunoconjugate can be administered to a non-human mammal expressingTAT226 with which the antibody cross-reacts (e.g., a primate, pig, rat,or mouse) for veterinary purposes or as an animal model of humandisease. Regarding the latter, such animal models may be useful forevaluating the therapeutic efficacy of antibodies or immunoconjugates ofthe invention (e.g., testing of dosages and time courses ofadministration).

Antibodies or immunoconjugates of the invention can be used either aloneor in combination with other compositions in a therapy. For instance, anantibody or immunoconjugate of the invention may be co-administered withat least one additional therapeutic agent and/or adjuvant. In certainembodiments, an additional therapeutic agent is a cytotoxic agent, achemotherapeutic agent, or a growth inhibitory agent. In one of suchembodiments, a chemotherapeutic agent is an agent or a combination ofagents used in the treatment of ovarian cancer, such as a platinumcompound (e.g., cisplatin or carboplatin); a taxane (e.g., paclitaxel ordocetaxel); topotecan; an anthracycline (e.g., doxorubicin (ADRIAMYCIN®)or liposomal doxorubicin (DOXIL®)); gemcitabine; cyclophosphamide;vinorelbine (NAVELBINE®); hexamethylmelamine; ifosfamide; and etoposide.In another of such embodiments, a chemotherapeutic agent is an agent ora combination of agents used in the treatment of uterine or endometrialcancer, such as cisplatin, carboplatin, doxorubicin, paclitaxel,methotrexate, fluorouracil and medroxyprogesterone. In another of suchembodiments, a chemotherapeutic agent is an agent or combination ofagents used in the treatment of brain tumors, such as a nitrosourea(e.g., carmustine or lomustine) a cytotoxic agen (e.g., irinotecan ortemozolamide); an anti-angiogenic agent (e.g., thalidomide, TNP-470,platelet factor 4, interferon and endostatin); a differentiating agent(e.g., retinoids, phenylbutyrate, phenylacetate, and anti-neoplastons);an anti-invasion agent (e.g., matrix metalloproteinase inhibitors suchas marimastat); a signal transduction modulator (e.g., tamoxifen,bryostatin, and O-6 benzyguanine); a topoisomerase inhibitor (e.g.,irinotecan or topotecan); and a growth factor inhibitor (e.g., atyrosine kinase inhibitor). In another of such embodiments, achemotherapeutic agent is an agent or a combination of agents used inthe treatment of kidney cancer (e.g., Wilms' tumor), such asvincristine, actinomycin D, adriamycin, doxorubicin, cyclophosphamide,ifosfamide, etoposide, and carboplatin. In certain embodiments, anantibody of the invention may be combined with an anti-inflammatoryand/or antiseptic.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody or immunoconjugate of the invention canoccur prior to, simultaneously, and/or following, administration of theadditional therapeutic agent and/or adjuvant. Antibodies orimmunoconjugates of the invention can also be used in combination withradiation therapy.

An antibody or immunoconjugate of the invention (and any additionaltherapeutic agent or adjuvant) can be administered by any suitablemeans, including parenteral, subcutaneous, intraperitoneal,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, the antibody orimmunoconjugate is suitably administered by pulse infusion, particularlywith declining doses of the antibody or immunoconjugate. Dosing can beby any suitable route, e.g. by injections, such as intravenous orsubcutaneous injections, depending in part on whether the administrationis brief or chronic.

When the binding target is located in the brain, certain embodiments ofthe invention provide for the antibody or immunoconjugate to traversethe blood-brain barrier. Several art-known approaches exist fortransporting molecules across the blood-brain barrier, including, butnot limited to, physical methods, lipid-based methods, stem cell-basedmethods, and receptor and channel-based methods.

Physical methods of transporting an antibody or immunoconjugate acrossthe blood-brain barrier include, but are not limited to, circumventingthe blood-brain barrier entirely, or by creating openings in theblood-brain barrier. Circumvention methods include, but are not limitedto, direct injection into the brain (see, e.g., Papanastassiou et al.,Gene Therapy 9: 398-406 (2002)), interstitialinfusion/convection-enhanced delivery (see, e.g., Bobo et al., Proc.Natl. Acad. Sci. USA 91: 2076-2080 (1994)), and implanting a deliverydevice in the brain (see, e.g., Gill et al., Nature Med. 9: 589-595(2003); and Gliadel Wafers™, Guildford Pharmaceutical). Methods ofcreating openings in the barrier include, but are not limited to,ultrasound (see, e.g., U.S. Patent Publication No. 2002/0038086),osmotic pressure (e.g., by administration of hypertonic mannitol(Neuwelt, E. A., Implication of the Blood-Brain Barrier and itsManipulation, Vols 1 & 2, Plenum Press, N.Y. (1989)), permeabilizationby, e.g., bradykinin or permeabilizer A-7 (see, e.g., U.S. Pat. Nos.5,112,596, 5,268,164, 5,506,206, and 5,686,416), and transfection ofneurons that straddle the blood-brain barrier with vectors containinggenes encoding the antibody (see, e.g., U.S. Patent Publication No.2003/0083299).

Lipid-based methods of transporting an antibody or immunoconjugateacross the blood-brain barrier include, but are not limited to,encapsulating the antibody or immunoconjugate in liposomes that arecoupled to antibody binding fragments that bind to receptors on thevascular endothelium of the blood-brain barrier (see, e.g., U.S. PatentApplication Publication No. 20020025313), and coating the antibody orimmunoconjugate in low-density lipoprotein particles (see, e.g., U.S.Patent Application Publication No. 20040204354) or apolipoprotein E(see, e.g., U.S. Patent Application Publication No. 20040131692).

Stem-cell based methods of transporting an antibody or immunoconjugateacross the blood-brain barrier entail genetically engineering neuralprogenitor cells (NPCs) to express the antibody or immunoconjugate ofinterest and then implanting the stem cells into the brain of theindividual to be treated. See Behrstock et al. (2005) Gene Ther. 15 Dec.2005 advanced online publication (reporting that NPCs geneticallyengineered to express the neurotrophic factor GDNF reduced symptoms ofParkinson disease when implanted into the brains of rodent and primatemodels).

Receptor and channel-based methods of transporting an antibody orimmunoconjugate across the blood-brain barrier include, but are notlimited to, using glucocorticoid blockers to increase permeability ofthe blood-brain barrier (see, e.g., U.S. Patent Application PublicationNos. 2002/0065259, 2003/0162695, and 2005/0124533); activating potassiumchannels (see, e.g., U.S. Patent Application Publication No.2005/0089473), inhibiting ABC drug transporters (see, e.g., U.S. PatentApplication Publication No. 2003/0073713); coating antibodies orimmunoconjugates with a transferrin and modulating activity of the oneor more transferrin receptors (see, e.g., U.S. Patent ApplicationPublication No. 2003/0129186), and cationizing the antibodies orimmunoconjugates (see, e.g., U.S. Pat. No. 5,004,697).

Antibodies or immunoconjugates of the invention would be formulated,dosed, and administered in a fashion consistent with good medicalpractice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The antibody or immunoconjugate need notbe, but is optionally formulated with one or more agents currently usedto prevent or treat the disorder in question. The effective amount ofsuch other agents depends on the amount of antibody or immunoconjugatepresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody or immunoconjugate of the invention (when used alone or incombination with one or more other additional therapeutic agents, suchas chemotherapeutic agents) will depend on the type of disease to betreated, the type of antibody or immunoconjugate, the severity andcourse of the disease, whether the antibody or immunoconjugate isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody orimmunoconjugate, and the discretion of the attending physician. Theantibody or immunoconjugate is suitably administered to the patient atone time or over a series of treatments. Depending on the type andseverity of the disease, about 1 mg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10mg/kg) of antibody or immunoconjugate can be an initial candidate dosagefor administration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. One typical dailydosage might range from about 1 mg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment wouldgenerally be sustained until a desired suppression of disease symptomsoccurs. One exemplary dosage of the antibody or immunoconjugate would bein the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or moredoses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or anycombination thereof) may be administered to the patient. Such doses maybe administered intermittently, e.g. every week or every three weeks(e.g. such that the patient receives from about two to about twenty, ore.g. about six doses of the antibody or immunoconjugate). An initialhigher loading dose, followed by one or more lower doses may beadministered. An exemplary dosing regimen comprises administering aninitial loading dose of about 4 mg/kg, followed by a weekly maintenancedose of about 2 mg/kg of the antibody. However, other dosage regimensmay be useful. The progress of this therapy is easily monitored byconventional techniques and assays.

3. Assays

Anti-TAT226 antibodies and immunoconjugates of the invention may becharacterized for their physical/chemical properties and/or biologicalactivities by various assays known in the art.

a) Activity Assays

In one aspect, assays are provided for identifying anti-TAT226antibodies or immunoconjugates thereof having biological activity.Biological activity may include, e.g., the ability to inhibit cellgrowth or proliferation (e.g., “cell killing” activity), or the abilityto induce cell death, including programmed cell death (apoptosis).Antibodies or immunoconjugates having such biological activity in vivoand/or in vitro are also provided.

In certain embodiments, an anti-TAT226 antibody or immunoconjugatethereof is tested for its ability to inhibit cell growth orproliferation in vitro. Assays for inhibition of cell growth orproliferation are well known in the art. Certain assays for cellproliferation, exemplified by the “cell killing” assays describedherein, measure cell viability. One such assay is the CellTiter-Glo™Luminescent Cell Viability Assay, which is commercially available fromPromega (Madison, Wis.). That assay determines the number of viablecells in culture based on quantitation of ATP present, which is anindication of metabolically active cells. See Crouch et al (1993) J.Immunol. Meth. 160:81-88, U.S. Pat. No. 6,602,677. The assay may beconducted in 96- or 384-well format, making it amenable to automatedhigh-throughput screening (HTS). See Cree et al (1995) AntiCancer Drugs6:398-404. The assay procedure involves adding a single reagent(CellTiter-Glo® Reagent) directly to cultured cells. This results incell lysis and generation of a luminescent signal produced by aluciferase reaction. The luminescent signal is proportional to theamount of ATP present, which is directly proportional to the number ofviable cells present in culture. Data can be recorded by luminometer orCCD camera imaging device. The luminescence output is expressed asrelative light units (RLU).

Another assay for cell proliferation is the “MTT” assay, a colorimetricassay that measures the oxidation of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazanby mitochondrial reductase. Like the CellTiter-Glo™ assay, this assayindicates the number of metabolically active cells present in a cellculture. See, e.g., Mosmann (1983) J. Immunol. Meth. 65:55-63, and Zhanget al. (2005) Cancer Res. 65:3877-3882.

In one aspect, an anti-TAT226 antibody is tested for its ability toinduce cell death in vitro. Assays for induction of cell death are wellknown in the art. In some embodiments, such assays measure, e.g., lossof membrane integrity as indicated by uptake of propidium iodide (PI),trypan blue (see Moore et al. (1995) Cytotechnology, 17:1-11), or 7AAD.In an exemplary PI uptake assay, cells are cultured in Dulbecco'sModified Eagle Medium (D-MEM):Ham's F-12 (50:50) supplemented with 10%heat-inactivated FBS (Hyclone) and 2 mM L-glutamine. Thus, the assay isperformed in the absence of complement and immune effector cells. Cellsare seeded at a density of 3×10⁶ per dish in 100×20 mm dishes andallowed to attach overnight. The medium is removed and replaced withfresh medium alone or medium containing various concentrations of theantibody or immunoconjugate. The cells are incubated for a 3-day timeperiod. Following treatment, monolayers are washed with PBS and detachedby trypsinization. Cells are then centrifuged at 1200 rpm for 5 minutesat 4° C., the pellet resuspended in 3 ml cold Ca²⁺ binding buffer (10 mMHepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂) and aliquoted into 35 mmstrainer-capped 12×75 mm tubes (1 ml per tube, 3 tubes per treatmentgroup) for removal of cell clumps. Tubes then receive PI (10 μg/ml).Samples are analyzed using a FACSCAN™ flow cytometer and FACSCONVERT™CellQuest software (Becton Dickinson). Antibodies or immunoconjugateswhich induce statistically significant levels of cell death asdetermined by PI uptake are thus identified.

In one aspect, an anti-TAT226 antibody or immunoconjugate is tested forits ability to induce apoptosis (programmed cell death) in vitro. Anexemplary assay for antibodies or immunconjugates that induce apoptosisis an annexin binding assay. In an exemplary annexin binding assay,cells are cultured and seeded in dishes as discussed in the precedingparagraph. The medium is removed and replaced with fresh medium alone ormedium containing 0.001 to 10 μg/ml of the antibody or immunoconjugate.Following a three-day incubation period, monolayers are washed with PBSand detached by trypsinization. Cells are then centrifuged, resuspendedin Ca²⁺ binding buffer, and aliquoted into tubes as discussed in thepreceding paragraph. Tubes then receive labeled annexin (e.g. annexinV-FITC) (1 μg/ml). Samples are analyzed using a FACSCAN™ flow cytometerand FACSCONVERT™ CellQuest software (BD Biosciences). Antibodies orimmunoconjugates that induce statistically significant levels of annexinbinding relative to control are thus identified. Another exemplary assayfor antibodies or immunconjugates that induce apoptosis is a histone DNAELISA colorimetric assay for detecting internucleosomal degradation ofgenomic DNA. Such an assay can be performed using, e.g., the Cell DeathDetection ELISA kit (Roche, Palo Alto, Calif.).

Cells for use in any of the above in vitro assays include cells or celllines that naturally express TAT226 or that have been engineered toexpress TAT226. Such cells include tumor cells that overexpress TAT226relative to normal cells of the same tissue origin. Such cells alsoinclude cell lines (including tumor cell lines) that express TAT226 andcell lines that do not normally express TAT226 but have been transfectedwith nucleic acid encoding TAT226. Exemplary cell lines provided hereinfor use in any of the above in vitro assays include the OVCAR3 humanovarian cancer cell line, which expresses TAT226, and the HCT116 humancolon cancer cell line transfected with nucleic acid encoding TAT226.

In one aspect, an anti-TAT226 antibody or immunoconjugate thereof istested for its ability to inhibit cell growth or proliferation in vivo.In certain embodiments, an anti-TAT226 antibody or immunoconjugatethereof is tested for its ability to inhibit tumor growth in vivo. Invivo model systems, such as xenograft models, can be used for suchtesting. In an exemplary xenograft system, human tumor cells areintroduced into a suitably immunocompromised non-human animal, e.g., anathymic “nude” mouse. An antibody or immunoconjugate of the invention isadministered to the animal. The ability of the antibody orimmunoconjugate to inhibit or decrease tumor growth is measured. Incertain embodiments of the above xenograft system, the human tumor cellsare tumor cells from a human patient. Such xenograft models arecommercially available from Oncotest GmbH (Frieberg, Germany). Incertain embodiments, the human tumor cells are cells from a human tumorcell line, such as OVCAR3 cells, as exemplified herein. In certainembodiments, the human tumor cells are introduced into a suitablyimmunocompromised non-human animal by subcutaneous injection or bytransplantation into a suitable site, such as a mammary fat pad.

b) Binding Assays and Other Assays

In one aspect, an anti-TAT226 antibody is tested for its antigen bindingactivity. For example, in certain embodiments, an anti-TAT226 antibodyis tested for its ability to bind to TAT226 expressed on the surface ofa cell. A FACS assay such as that described in Example D may be used forsuch testing.

In one aspect, competition assays may be used to identify a monoclonalantibody that competes with YWO.32, YWO.49, YWO.49.B7, YWO.49.C9,YWO.49.H2, or YWO.49.H6 for binding to TAT226. In certain embodiments,such a competing antibody binds to the same epitope (e.g., a linear or aconformational epitope) that is bound by YWO.32, YWO.49, YWO.49.B7,YWO.49.C9, YWO.49.H2, or YWO.49.H6. Exemplary competition assaysinclude, but are not limited to, routine assays such as those providedin Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.). Detailed exemplarymethods for mapping an epitope to which an antibody binds are providedin Morris (1996) “Epitope Mapping Protocols,” in Methods in MolecularBiology vol. 66 (Humana Press, Totowa, N.J.). Two antibodies are said tobind to the same epitope if each blocks binding of the other by 50% ormore.

In an exemplary competition assay, immobilized TAT226 is incubated in asolution comprising a first labeled antibody that binds to TAT226 (e.g.,YWO.32, YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2, or YWO.49.H6) and asecond unlabeled antibody that is being tested for its ability tocompete with the first antibody for binding to TAT226. The secondantibody may be present in a hybridoma supernatant. As a control,immobilized TAT226 is incubated in a solution comprising the firstlabeled antibody but not the second unlabeled antibody. After incubationunder conditions permissive for binding of the first antibody to TAT226,excess unbound antibody is removed, and the amount of label associatedwith immobilized TAT226 is measured. If the amount of label associatedwith immobilized TAT226 is substantially reduced in the test samplerelative to the control sample, then that indicates that the secondantibody is competing with the first antibody for binding to TAT226. Incertain embodiments, immobilized TAT226 is present on the surface of acell or in a membrane preparation obtained from a cell expressing TAT226on its surface.

In one aspect, purified anti-TAT226 antibodies can be furthercharacterized by a series of assays including, but not limited to,N-terminal sequencing, amino acid analysis, non-denaturing sizeexclusion high pressure liquid chromatography (HPLC), mass spectrometry,ion exchange chromatography and papain digestion.

In one embodiment, the invention contemplates an altered antibody thatpossesses some but not all effector functions, which make it a desirablecandidate for many applications in which the half life of the antibodyin vivo is important yet certain effector functions (such as complementand ADCC) are unnecessary or deleterious. In certain embodiments, the Fcactivities of the antibody are measured to ensure that only the desiredproperties are maintained. In vitro and/or in vivo cytotoxicity assayscan be conducted to confirm the reduction/depletion of CDC and/or ADCCactivities. For example, Fc receptor (FcR) binding assays can beconducted to ensure that the antibody lacks FcγR binding (hence likelylacking ADCC activity), but retains FcRn binding ability. The primarycells for mediating ADCC, NK cells, express Fc(RIII only, whereasmonocytes express Fc(RI, Fc(RII and Fc(RIII. FcR expression onhematopoietic cells is summarized in Table 3 on page 464 of Ravetch andKinet, Annu. Rev. Immunol. 9:457-92 (1991). An example of an in vitroassay to assess ADCC activity of a molecule of interest is described inU.S. Pat. No. 5,500,362 or 5,821,337. Useful effector cells for suchassays include peripheral blood mononuclear cells (PBMC) and NaturalKiller (NK) cells. Alternatively, or additionally, ADCC activity of themolecule of interest may be assessed in vivo, e.g., in a animal modelsuch as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).C1q binding assays may also be carried out to confirm that the antibodyis unable to bind C1q and hence lacks CDC activity. To assess complementactivation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996), may be performed. FcRn binding and invivo clearance/half life determinations can also be performed usingmethods known in the art.

F. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, etc. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is by itself or combined with another composition effective fortreating, preventing and/or diagnosing the condition and may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). At least one active agent in the composition is anantibody or immunoconjugate of the invention. The label or packageinsert indicates that the composition is used for treating the conditionof choice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody or immunoconjugate of the invention; and (b) asecond container with a composition contained therein, wherein thecomposition comprises a further cytotoxic or otherwise therapeuticagent. The article of manufacture in this embodiment of the inventionmay further comprise a package insert indicating that the compositionscan be used to treat a particular condition. Alternatively, oradditionally, the article of manufacture may further comprise a second(or third) container comprising a pharmaceutically-acceptable buffer,such as bacteriostatic water for injection (BWFI), phosphate-bufferedsaline, Ringer's solution and dextrose solution. It may further includeother materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, and syringes.

IV. EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

A. Analysis of TAT226 Gene Expression

Human TAT226 gene expression was analyzed using a proprietary databasecontaining gene expression information (GeneExpress®, Gene Logic Inc.,Gaithersburg, Md.). Graphical analysis of the GeneExpress® database wasconducted using a microarray profile viewer. FIG. 13 is a graphicrepresentation of TAT226 gene expression in various tissues, which arelisted at left. The scale across the top of the graph indicates geneexpression levels based on hybridization signal intensity. Dots appearboth above and below the line adjacent to each listed tissue. The dotsappearing above the line represent gene expression in normal tissue, andthe dots appearing below the line represent gene expression in tumor ordiseased tissue. FIG. 13 shows a trend toward increased TAT226 geneexpression in tumor or diseased tissues relative to their normalcounterparts. In particular, TAT226 shows substantial overexpression intumorous and diseased ovary relative to normal ovary and in Wilms' tumorrelative to normal kidney. Other tissues showing overexpression in tumoror diseased tissue relative to normal tissue include endometrial,adrenal, bone, lung, skin, and soft tissue. In addition, TAT226 isstrongly expressed in normal brain tissue (such as rhinencephalon,hippocampus, and basal ganglia) and in tumorous or diseased braintissue, such as gliomas.

The GeneExpress® database was also used to analyze human TAT226 geneexpression in normal ovary; normal fallopian tube; ovarian cancer of theclear cell, mucinous, and serous cystoadenocarcinoma subtypes;metastatic ovarian cancer; and other types of ovarian cancer. Theresults are reported graphically in FIG. 14, with the particular tissuetypes indicated underneath the graph. The scale on the y-axis of thegraph indicates gene expression levels based on hybridization signalintensity. Serous cystoadenocarcinoma and metastatic ovarian cancershowed strong overexpression of TAT226 relative to normal ovary. Clearcell and mucinous subtypes showed expression comparable to normal ovary.Normal fallopian tube also showed substantial expression of TAT226.Notably, the serous subtype of ovarian cancer closely resemblesfallopian tube epithelium histologically, and both ovary and fallopiantubes are derived from the same embryonic tissue. See Fox et al. (2002)“Pathology of epithelial ovarian cancer,” in Ovarian Cancer ch. 9(Jacobs et al., eds., Oxford University Press, New York)

B. Generation of Anti-TAT226 Antibodies

Antibodies to TAT226 were generated by screening a phage display librarywith a recombinant “TAT226-His” fusion protein comprising amino acids1-115 of SEQ ID NO:75 and a C-terminal polyhistidine tag. The phagedisplay library was a synthetic (Fab“)₂ library generated using aFab”-zip-phage system. See Lee et al. (2004) J. Immunol. Methods284:119-132. The library comprised a library of heavy chain HVRs in theframework of the huMAb4D5-8 heavy chain variable region (see FIGS. 5Aand 5B, second acceptor “B,” SEQ ID NOs:50, 51, 57, 35) and a fixedhuMAb4D5-8 light chain variable region as shown in SEQ ID NO:26. Clonesselected using phage display were screened against TAT226-His usingphage ELISA (see, e.g., Sidhu et al. (2004) J. Mol. Biol. 338:299-310).Clones YWO.32 and YWO.49 were selected for further analysis.

To improve the affinity of YWO.49, phage display libraries weregenerated in the background of YWO.49, with HVR-H3 and HVR-L3 targetedfor soft-randomization, in which selected amino acid residues in thedesignated HVRs were held constant while others were subjected tomutagenesis. Selected clones were screened by phage ELISA. Affinitymatured antibodies designated YWO.49.B7, YWO.49.C9, YWO.49.H2, andYWO.49.H6 were selected for further analysis. The nucleotide and encodedpolypeptide sequences of the VH and VL regions of YWO.32, YWO.49,YWO.49.B7, YWO.49.C9, YWO.49.H2, and YWO.49.H6 were determined, as shownin FIGS. 9 and 10. The heavy and light chain HVR sequences of YWO.32,YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2, and YWO.49.H6 are shown inFIGS. 2-4. Consensus HVR-H3 and HVR-L3 sequences derived from YWO.49,YWO.49.B7, YWO.49.C9, YWO.49.H2, and YWO.49.H6 are also shown in FIG. 4.YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2, and YWO.49.H6 were“reformatted” as full-length IgGs by grafting Fab′ fragments ontoappropriate constant regions using recombinant techniques. Theexperiments described hereinafter were performed using the reformattedantibodies.

C. Characterization of Binding Affinity to Recombinant Antigen

The binding affinity of YWO.49, YWO.49.B7, YWO.49.C9, YWO.49.H2, andYWO.49.H6 for recombinant antigen was determined by surface plasmonresonance measurement using a BIACORE® 3000 system (Biacore, Inc.,Piscataway, N.J.). Briefly, carboxymethylated dextran biosensor chips(CMS, Biacore Inc.) were activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Anti-TAT226 antibody was diluted to 5 ug/ml with 10 mM sodium acetate,pH 4.8, before injection at a flow rate of 5 ul/minute to achieveapproximately 500 response units (RU) of coupled antibody. Next, 1Methanolamine was injected to block unreacted groups. For kineticsmeasurements, two-fold serial dilutions of TAT226-His (0.7 nM to 500 nM)were injected in PBS with 0.05% Tween 20 at 25° C. at a flow rate of 25μl/min. Association rates (k_(on)) and dissociation rates (k_(off)) werecalculated using a simple one-to-one Langmuir binding model(BIAevaluation Software version 3.2). The equilibrium dissociationconstant (Kd) was calculated as the ratio k_(off)/k_(on). The results ofthis experiment are shown in Table 2 below.

TABLE 2 Clone k_(on)/10⁵ k_(off)/10⁻⁴ Kd (nM) YWO.49 0.074 3.49 47.16YWO.49.B7 0.27 <0.05 <0.18 YWO.49.C9 1.53 <0.05 <0.03 YWO.49.H2 0.220.05 0.23 YWO.49.H6 1.86 0.09 0.05

D. Characterization of Antibody Binding to Cell Surface TAT226

The ability of anti-TAT226 antibodies to bind to TAT226 expressed on thesurface of OVCAR3, a human ovarian cancer cell line, was examined.Fluorescence-activated cell sorting (FACS) was performed on OVCAR3 cellsin the absence and in the presence of YWO.49, YWO.49.B7, YWO.49.C9,YWO.49.H2, or YWO.49.H6. Briefly, detached cells were incubated with 5μg/ml primary antibody for 1 hour on ice, washed, and incubated withsecondary antibody (anti-human IgG conjugated to phycoerythrin) for 30minutes on ice. FACS was performed using a FACScan™ flow cytometer (BDBiosciences, San Jose, Calif.).

The results of FACS analysis for YWO.49, YWO.49.H2, and YWO.H6 are shownin FIG. 15. The peaks on the left side of each graph represent“background” binding, i.e., binding of secondary antibody only. Thepeaks on the right side of each graph represent binding of the indicatedanti-TAT226 antibody. YWO.49.B7 did not bind significantly to OVCAR3cells by FACS, even though it bound to recombinant antigen with a Kdwithin the range of Kds observed for YWO.49 and the other affinitymatured antibodies (see results of BIACORE® analysis, table 2 above).Binding of YWO.49.C9 was comparable to that observed for YWO.49.H2 andYWO.H6.

E. Characterization of Binding Affinity to Cell Surface Antigen

The binding affinity of YWO.49.H2 and YWO.49.H6 for TAT226 expressed onthe surface of OVCAR3 cells was examined using a competition assay.Briefly, labeled (iodinated) YWO.49.H2 or YWO.49.H6 was allowed to bindto OVCAR3 cells in the presence of unlabeled antibody. Binding affinityof the antibodies was determined in accordance with the Scatchardanalysis methodology initially described in Munson et al., Anal.Biochem. 107:220 (1980). The results of this experiment are shown inTable 3 below.

TABLE 3 Clone Kd (nM) YWO.49.H2 0.348 YWO.49.H6 0.404The Kd of YWO.49.H2 and YWO.49.H6 is higher for TAT226 expressed on thesurface of OVCAR3 cells compared with recombinant TAT226-H1s (compareKds for YWO.49.H2 and YWO.49.H6 in tables 2 and 3), indicating thatYWO.49.H2 and YWO.49.H6 bind with slightly higher affinity torecombinant TAT226-His than to TAT226 expressed on the surface of OVCAR3cells.

F. TAT226 mRNA and Protein Expression

TAT226 mRNA expression in OVCAR3 cells and in a panel of ovarian cancersamples was analyzed using a 5′ nuclease (TaqMan®) assay and real-timequantitative PCR. The ovarian cancer samples, designated “HF ####” inFIG. 16, were frozen tissue sections. RNA was isolated from the tissuesections, amplified using Ambion's Message Amp II kit (Ambion, Austin,Tex.), and reverse transcribed into cDNA. RNA was isolated from OVCAR3cells and reverse-transcribed into cDNA. TAT226 cDNA was amplified byreal-time PCR in the presence of a non-extendible, reporter probespecific for the amplification product. The threshold cycle or “Ct” (thecycle in which the signal generated from cleavage of the reporter probeexceeded background) was determined and used to calculate startingTAT226 mRNA levels. The level of TAT226 mRNA in the panel of ovariancancer samples was expressed relative to the level of TAT226 mRNA inOVCAR3 cells, as shown in the bar graph in FIG. 16.

TAT226 protein expression in OVCAR3 cells and in the above panel ofovarian cancer samples was analyzed using immunohistochemistry (IHC) asfollows. Tissue sections (frozen or paraffin-embedded) of the ovariancancer samples were fixed for 5 minutes in acetone/ethanol. The sectionswere washed in PBS, blocked with avidin and biotin (Vector Laboratories,Inc., Burlingame, Calif.) for 10 minutes each, and washed in PBS again.The sections were then blocked with 10% serum for 20 minutes and blottedto remove excess serum. Primary antibody (YWO.49.H2 or YWO.49.H6) wasthen added to the sections at a concentration of 10 μg/ml for 1 hour.The sections were then washed in PBS. Biotinylated secondary anti-humanantibody was added to the sections for 30 minutes, and then the sectionswere washed with PBS. The sections were then exposed to the reagents ofthe Vector ABC kit (Vector Laboratories, Inc., Burlingame, Calif.) for30 minutes and then washed in PBS. The sections were then exposed todiaminobenzidine (Pierce) for 5 minutes and then washed in PBS. Thesections were then counterstained with Mayers hematoxylin, covered witha coverslip, and visualized. IHC was performed on OVCAR3 cells using thesame protocol, except that the cells were first pelleted, frozen, andthen sectioned. The sections were then subjected to the above protocol.

The results are reported qualitatively in FIG. 16, with expressionlevels categorized as “−”, “+/−”, or “+”. Generally, there was anoverall correlation between the level of TAT226 mRNA expression and theexpression of TAT226 protein on the surface of OVCAR3 cells. The IHCexperiments also confirmed that the antibodies recognized TAT226 on thecell surface. The histology of each cell in the ovarian cancer cellpanel is also reported in FIG. 16, with the abbreviation “adenoca.”denoting “adenocarcinoma.”

G. Production of Anti-TAT226 ADCs

Anti-TAT226 ADCs were produced by conjugating YWO.49.H2 and YWO.49.H6 tothe following drug-linker moieties: MC-vc-PAB-MMAE; MC-vc-PAB-MMAF; andMC-MMAF, which are depicted above in Section III.C.1.b.2. Prior toconjugation, the antibodies were partially reduced with TCEP usingstandard methods in accordance with the methodology described in WO2004/010957 A2. The partially reduced antibodies were conjugated to theabove drug-linker moieties using standard methods in accordance with themethodology described in Doronina et al. (2003) Nat. Biotechnol.21:778-784 and US 2005/0238649 A1. Briefly, the partially reducedantibodies were combined with the drug linker moieties to allowconjugation of the moieties to cysteine residues. The conjugationreactions were quenched, and the ADCs were purified. The drug load(average number of drug moieties per antibody) for each ADC wasdetermined by HPLC, as follows:

ADC Drug load YWO.49.H2-MC-vc-PAB-MMAE 3.8 YWO.49.H2-MC-vc-PAB-MMAF 4.7YWO.49.H2-MC-MMAF 4.9 YWO.49.H6-MC-vc-PAB-MMAE 4.4YWO.49.H6-MC-vc-PAB-MMAF 4.4 YWO.49.H6-MC-MMAF 4.1

H. Cell Killing Assays

Antibody-drug conjugates (ADCs) were tested for the ability to inhibitproliferation of cells expressing TAT226 in the following in vitro andin vivo cell killing assays:

1. OVCAR3 In Vitro Cell Killing Assay

YWO.49.H2 and YWO.49.H6 ADCs were tested for the ability to inhibitproliferation of OVCAR3 cells. OVCAR3 cells were seeded in 96-wellplates in RPMI with 20% FBS. OVCAR3 cells at a density of 3000 cells perwell were incubated with varying concentrations of the ADCs, as shown inFIG. 17. Anti-MUC16/CA125 antibody conjugated to MC-vc-PAB-MMAE was usedas a positive control. MUC16/CA125 is a known ovarian cancer antigen.See, e.g., Yin et al. (2001) J. Biol. Chem. 276:27371-27375. Anti-IL-8antibody conjugated to MC-vc-PAB-MMAE was used as a negative control.After 5 days of incubation, cell viability was measured using theCellTiter-Glo™ Luminescent Cell Viability Assay (Promega, Madison, Wis.)according to the manufacturer's instructions. The scale on the y-axis ofFIG. 17 indicates the relative light units, or “RLUs,” from luciferaseluminescence, which is a measure of cell viability.

FIG. 17 shows that, similar to the positive control,YWO.49.H2-MC-vc-PAB-MMAF and YWO.49.H6-MC-vc-PAB-MMAF had marked cellkilling activity, particularly at concentrations at and around 0.01 and0.1 μg/ml. YWO.49.H2-MC-vc-PAB-MMAE and YWO.49.H6-MC-vc-PAB-MMAE alsohad cell killing activity, but to a lesser extent than seen withYWO.49.H2-MC-vc-PAB-MMAF and YWO.49.H6-MC-vc-PAB-MMAF. The IC₅₀ forYWO.49.H2-MC-vc-PAB-MMAF and YWO.49.H6-MC-vc-PAB-MMAF was about 0.005nM, and the IC₅₀ for YWO.49.H2-MC-vc-PAB-MMAE andYWO.49.H6-MC-vc-PAB-MMAE was about 0.2 nM. The IC₅₀ for free MMAE wasabout 0.1 nM. YWO.49.H2-MC-MMAF and YWO.49.H6-MC-MMAF did notdemonstrate significant cell killing activity in this assay. In thisparticular assay system, it is noted that at high concentrations of ADC,including negative control ADC, cell viability decreases substantiallydue to the overall high concentration of MMAE and MMAF.

2. In Vitro Cell Killing Assay Using HCT116 Transfected Cells

YWO.49.H2 and YWO.49.H6 ADCs were tested for the ability to inhibitproliferation of HCT116 cells, a colon cancer cell line, which had beenstably transfected with nucleic acid encoding human TAT226.Untransfected HCT116 cells are normally about 5-6 fold less sensitive tofree (unconjugated) MMAE than OVCAR3 cells.

Briefly, HCT116 cells were transfected as follows. Nucleic acid encodingepitope-tagged human TAT226 was constructed in the mammalian expressionvector pcDNA3.1 (Invitrogen, Carlsbad, Calif.). The epitope tagconsisted of amino acids 1-53 of the herpes simplex virus type 1glycoprotein D (the “gD” tag), which replaced the signal sequence fromamino acids 1-22 at the N-terminus of human TAT226. The recombinantvector was transfected into HCT116 cells using Lipofectamine200(Invitrogen) according to the manufacturer's protocol. TransfectedHCT116 cells were cultured in McCoy's 5a medium with 10% FBS and 0.4mg/ml G418. Cells were stained using anti-gD antibodies and sorted byFACS to select for individual clones expressing recombinant gD:humanTAT226 fusion protein. One of the clones, designated HCT116#9-4, wasselected for further analysis.

To perform the cell killing assay, HCT116#9-4 cells were seeded in96-well plates. HCT116#9-4 cells at a density of 1000 cells per wellwere incubated with varying concentrations of the ADCs, as shown in FIG.18. Anti-gp120 antibody conjugated to MC-vc-PAB-MMAE was used as anegative control. After 3 days of incubation, cell viability wasmeasured using the CellTiter-Glo™ Luminescent Cell Viability Assay(Promega, Madison, Wis.) according to the manufacturer's instructions.

FIG. 18 shows that YWO.49.H2-MC-vc-PAB-MMAF and YWO.49.H6-MC-vc-PAB-MMAFhad marked cell killing activity, particularly at about 0.01 μg/ml andup to the highest concentrations tested. The IC₅₀ forYWO.49.H2-MC-vc-PAB-MMAF and YWO.49.H6-MC-vc-PAB-MMAF was about 0.05 nM,and the IC₅₀ for free MMAE was about 0.9 nM. YWO.49.H2-MC-vc-PAB-MMAEand YWO.49.H6-MC-vc-PAB-MMAE did not have substantial cell killingactivity relative to the negative control in this particular assay. Thedifference between the cell killing activity of YWO.49.H2-MC-vc-PAB-MMAEand YWO.49.H6-MC-vc-PAB-MMAE in this assay compared to the OVCAR3 cellkilling assay (above) may be attributable to a variety of factors, e.g.,cell density and/or differences in drug sensitivity.

It is noted that for some other cell lines tested that normally expressTAT226 mRNA or protein, YWO.49.H2 and YWO.49.H6 ADCs did not showsignificant cell killing activity. This could be due to a variety offactors, e.g., cell type-specific effects, levels of cell surface TAT226expression, and/or differences in drug sensitivity.

3. In Vivo Assay Using HCT116#9-4 Xenograft

An in vivo xenograft model was used to test unconjugated and conjugatedYWO.49.H6 for the ability to inhibit proliferation of TAT226-expressingtumor cells in vivo. Tumors were induced in athymic nude “nu-nu” mice bysubcutaneous injection of about 5×10⁶ HCT116#9-4 cells into the dorsalflanks of the mice. Tumors were allowed to grow until they reached amean tumor volume of 200 mm³. That point in time was designated as “day0.” As shown in FIG. 19, the mice received intravenous injections of 3mg/kg of the indicated unconjugated antibodies or ADCs on days 0, 7, and16. Unconjugated and conjugated anti-ragweed antibodies (Ab) served asnegative controls. Mean tumor volume was measured on days 3, 7, 10, 16,and 21. As shown in FIG. 19, YWO.49.H6-MC-vc-PAB-MMAF showed significanttumor cell killing activity, as measured by mean tumor volume, comparedto anti-ragweed Ab-MC-vc-PAB-MMAF in this particular xenograft model.YWO.49.H6-MC-vc-PAB-MMAE did not show significant tumor cell killingactivity relative to anti-ragweed Ab-MC-vc-PAB-MMAE in this xenograftmodel. However, this xenograft model may not reflect the cell killingactivity of YWO.49.H6-MC-vc-PAB-MMAE observed in vitro for a variety offactors, e.g., due to differences in drug sensitivity or expressionlevels of cell surface TAT226 in the xenograft tumor microenvironment.

4. Other Xenograft Models

Other xenograft models may be used to test unconjugated and conjugatedanti-TAT226 antibodies for the ability to inhibit proliferation ofTAT226-expressing tumor cells in vivo. For example, xenograft models forovarian and brain tumors may be provided by public sources such asOncotest GmbH (Frieberg, Germany) and Southern Research Institute(Birmingham, Ala.). The Oncotest models in particular were developed bygrowing patient tumors in immune-deficient nude mice. Xenografts thatexpress TAT226 mRNA and/or protein may be useful for demonstrating cellkilling activity of anti-TAT226 antibodies in vivo.

Conjugated YWO.49.H6 was tested in the Oncotest model OVXF1023 for theability to inhibit proliferation of ovarian tumor cells in vivo. TheOncotest model OVXF1023 is derived from a metastasized poorlydifferentiated papillary serous adenomatous ovarian carcinoma, stage M1.OVXF1023 mice were treated with the ADCs indicated in FIG. 20. In FIG.20, YWO.49.H6 is designated as “H6”; anti-ragweed (control) antibody isdesignated as “RW”; and the linker -MC-vc-PAB- is abbreviated as “vc.”The ADCs were administered on the days and at the concentrationsindicated in FIG. 20. The results shown in FIG. 20 indicate thatYWO.49.H6-MC-vc-PAB-MMAF and YWO.49.H6-MC-MMAF significantly reducedtumor volume relative to the other ADCs.

The above experiment was repeated with OVXF1023 under similar conditionsbut with changes in dosing and in the control ADCs. In the repeatexperiment, mice were treated with a higher dose (5 mg/kg) ofYWO.49.H6-MC-vc-PAB-MMAE and with lower doses (5 mg/kg) ofYWO.49.H6-MC-vc-PAB-MMAF and YWO.49.H6-MC-MMAF. The results demonstratedthat the H6 ADCs (and in particular YWO.49.H6-MC-vc-PAB-MMAE) showedreduced tumor volume relative to their respective control ADCs(anti-gp120-MC-vc-PAB-MMAE at 6.4 mg/kg; anti-gp120-MC-vc-PAB-MMAF at7.2 mg/kg; and anti-gp120-MC-MMAF at 5.4 mg/kg), although the differencein efficacy between the H6 ADCs and their respective control ADCs wasnot statistically significant for some data points. (Data not shown.)The difference between these results and the results obtained in thefirst OVXF1023 experiment may be attributable to the differences indosing of the H6 ADCs and the observation that the control anti-gp120ADCs showed unexpected activity in reducing tumor volume.

Additionally, the H6 ADCs were also tested in another Oncotest model,OVXF899, which is derived from a moderately differentiated papillaryserous ovarian carcinoma (primary tumor). The H6 ADCs did not reducetumor volume in this model. (Data not shown.) However, this particularOncotest model showed low expression of TAT226, which may account forthe observed results.

I. ThioMAbs

Cysteine engineered antibodies, or “thioMAbs,” were created in which aselected residue of YWO.49.H6 was substituted with cysteine in order toprovide an additional site for conjugation of a linker-drug moiety.Specifically, an A118C substitution (EU numbering) was made in the heavychain of YWO.49.H6, or a V205C substitution (Kabat numbering) was madein the light chain of YWO.49.H6. The resulting A118C thioMAb was thenconjugated to MC-MMAF, and the resulting V205C thioMAb was thenconjugated to either MC-MMAF or MC-vc-PAB-MMAE. All thioMAbs werecapable of binding to OVCAR3 cells by FACS analysis. (Data not shown.)

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literatures cited herein are expressly incorporated in theirentirety by reference.

What is claimed is:
 1. A method of treating a cell proliferativedisorder comprising cells that express TAT226, comprising administeringto an individual having said disorder an effective amount of an antibodythat binds to TAT226, or antigen-binding fragment thereof, wherein theantibody comprises: a heavy chain variable domain comprising a HVR-H1comprising the amino acid sequence of SEQ ID NO:4, a HVR-H2 comprisingthe amino acid sequence of SEQ ID NO:5, and a HVR-H3 comprising an aminoacid sequence selected from SEQ ID NO:6-10; a light chain variabledomain comprising a HVR-L1 comprising the amino acid sequence of SEQ IDNO:12, a HVR-L2 comprising the amino acid sequence of SEQ ID NO:13, anda HVR-L3 comprising an amino acid sequence selected from SEQ IDNO:14-18, wherein the antibody or antigen-binding fragment thereof iscovalently attached to a cytotoxic agent to form an antibody-drugconjugate.
 2. The method of claim 1, wherein the HVR-H3 comprises theamino acid sequence of SEQ ID NO:9, and the HVR-L3 comprises the aminoacid sequence of SEQ ID NO:17.
 3. The method of claim 1, wherein HVR-H3comprises the amino acid sequence of SEQ ID NO:10, and the HVR-L3comprises the amino acid sequence of SEQ ID NO:18.
 4. The method ofclaim 1, wherein said antibody or antigen-binding fragment thereoffurther comprises at least one framework selected from a VH subgroup IIIconsensus framework and a VL subgroup I consensus framework.
 5. Themethod of claim 1, wherein the antibody comprises a heavy chain variabledomain comprising an amino acid sequence selected from SEQ ID NO:21-25and a light chain variable domain comprising an amino acid sequenceselected from SEQ ID NO:26-31.
 6. The method of claim 5, wherein theheavy chain variable domain comprises the amino acid sequence of SEQ IDNO:24, and the light chain variable domain comprises the amino acidsequence of SEQ ID NO:29.
 7. The method of claim 5, wherein the heavychain variable domain comprises the amino acid sequence of SEQ ID NO:25,and the light chain variable domain comprises the amino acid sequence ofSEQ ID NO:30.
 8. The method of claim 1, wherein said antibody orantigen-binding fragment thereof is monoclonal.
 9. The method of claim1, wherein said antigen-binding fragment is selected from the groupconsisting of Fab, Fab′-SH, Fv, scFv, and (Fabl)₂ fragments.
 10. Themethod of claim 8, wherein said antibody or antigen-binding fragmentthereof is humanized.
 11. The method of any one of claims 1 to 7,wherein the cytotoxic agent is selected from the group consisting of atoxin, a chemotherapeutic agent, an antibiotic, a radioactive isotope,and a nucleolytic enzyme.
 12. The method of claim 1, wherein the cellproliferative disorder is selected from ovarian cancer, uterine cancer,brain tumor, and Wilms' tumor.
 13. The method of claim 12, wherein thecell proliferative disorder is associated with increased expression ofTAT226 on the surface of a cell.
 14. The method of any one of claims 1to 7, wherein the antibody is a bispecific antibody.
 15. The method ofany one of claims 1-7, wherein the antibody-drug conjugate comprises theformula Ab-(L-D)p, wherein (a) Ab is the antibody that binds to TAT226,or antigen-binding fragment thereof; (b) L is a linker; (c) D is a drug;and (d) p is 1 to about
 8. 16. The method of claim 15, wherein Lcomprises one or more of 6-maleimidocaproyl (“MC”), maleimidopropanoyl(“MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (a “PAB”), N-Succinimidyl4-(2-pyridylthio) pentanoate (“SPP”), N-succinimidyl4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“SMCC”), andN-Succinimidyl (4-iodo-acetyl) aminobenzoate (“SIAB”).
 17. The method ofclaim 15, wherein D comprises one or more of maytansinoid, auristatin,and dolastatin.
 18. The method of claim 16, wherein D comprises one ormore of maytansinoid, auristatin, and dolastatin.
 19. The method ofclaim 17, wherein D comprises auristatin and the auristatin is MMAE orMMAF.
 20. The method of claim 18, wherein D comprises auristatin and theauristatin is MMAE or MMAF.